DEVELOPMENTAL
BIOLOGY
Ontogeny
56, 426-430
of Tyrosine JORMA
Department
(1977)
Aminotransferase
J. OHISALO
of Medical
Chemistry, SF-001
Received
September
in Xenopus
AND JAAKKO
University 70 Helsinki 9,1976;
P. PISPA
of Helsinki, 17, Finland accepted
laevis
October
Siltavuorenpenger
10 A,
11,1976
The development of tyrosine aminotransferase (TAT) activity in Xenopus laevis embryos was studied. Undivided eggs can transaminate tyrosine to some extent. The enzyme activity increases after hatching on the third day of development. In the early stages of development, the transamination of tyrosine is due to aspartate aminotransferase (ASAT, EC 2.6.1.11, both isoenzymes of which are present in the undivided egg. No specific TAT (EC 2.6.1.5) can be detected until the age of about 1 day, at which time neurulation is complete and the rapid development of the foregut and visceral pouches and arches has begun. The appearance of the enzyme is immediately preceded by a steep increase in the concentration of free tyrosine. Tvrosine aminotransferase is known to be induced by its substrate in the adult liver, and a similar effect may operate in the embryo.
tyrosine aminotransferase during the embryonal development of the clawed toad, Xenopus laevis. Xenopus was chosen as the test animal because of the relative convenience of obtaining embryonal tissues in sufficient amounts and because the development of the embryo can be easily monitored visually.
INTRODUCTION
r.-Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5), TAT, is the enzyme mainly responsible for the catabolism of the amino acid tyrosine in the liver. In the rat the enzyme has very complicated hormonaI regulation (6, 8, 9, 12, 15, 20), while, in the frog, the enzyme activity has been shown to be affected only by tyrosine, dibutyryl cyclic AMP, and glucose (3, 16). The enzyme has several subforms, and the isoenzymes of aspartate aminotransferase (EC 2.6.1.1), ASAT, can be detected as “multiple forms” of tyrosine aminotransferase, due to their promiscuous substrate specificity (17). The mammalian enzyme is known to be inducible in the fetal liver (10) and in organ culture of fetal liver (13). The activity is increased at the time of birth and during the neonatal period in mammalian liver (11). The factors responsible for the increase are unknown, and the possibility that enzymes other than TAT might also transaminate tyrosine in vitro and hence cause false results has not been considered. The aim of the present work was to study the time of appearance of specific
MATERIALS
AND
METHODS
The frogs were a gift from Professor Sulo Toivonen from the Department of Zoology, University of Helsinki. They were housed, fed, and induced to lay eggs as advised by Nieuwkoop and Faber (14), whose normal tables were also used in staging. The staged embryos were counted and frozen immediately in liquid nitrogen. They were stored at -70°C. For assays, the embryos were allowed to thaw and were then homogenized in 3 vol of 0.5% Triton X-100. The homogenates were centrifuged at 10,OOOgfor 15 min, except for the electrofocusing experiments, for which they were centrifuged at 100,OOOgfor 60 min. After removing the fatty layer by aspiration, the supernatant fractions were used in the experiments. The isoelectric focusing was 426
Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
BRIEF
performed in a glycerol density gradient as described previously (17). The supernatant fractions from at least 500 embryos were divided equally between both gradient mixing vials. A pH gradient of 3.5-10 was generally used. The enzymes were assayed as described previously (17). Tyrosine was assayed by the method of Udenfriend and Cooper (19). The method of Lowry et al. (13) was used in protein estimations. The quantitative proportions of the different enzymes capable of transaminating tyrosine were estimated by drawing the isoelectric focusing profile on paper of even quality, cutting out the different peaks, and weighing them. The ampholines and the electrofocusing apparatus were obtained from LKB, Bromma, Sweden. The following reagents were used: L-tyrosine, pyridoxal-5’-phosphate (Sigma), ethylenediamine, glycerol, a-ketoglutaric acid (Merck), human chorionic gonadotropin (“Pregnyl,” Organon, Helsinki, Finland), and Triton X-100 (Koch-Light). The ASAT assay kit of Boehringer Mannheim, GmBH was used. RESULTS
AND
DISCUSSION
The development of total “tyrosine aminotransferase” activity is shown in Table 1. The initial activity is rather low, but an TABLE 1 TOTAL TYROSINE AMMINOTRANSFERASE ACTIVITY DURING THE EARLY DEVELOPMENT OF Xenopus laevis Stage
of development
TAT Picomoles of product per embryo per minute
1 18/19 23124 25126 27128 29/30 32 35/36 45146 47148
0.6 1.6 1.2 1.2 1.4 2.4 1.9 3.9 8.6 21.2
activity Picomoles of product per milligram of protein per minute 11 37 29 26 29 38 32 44 55 75
NOTES
427
increase is seen after stage 35136 i.e., at the age of 2.5 days. At this time, the walls of the primary hepatic cavity are folding up and starting to fill up the cavity. In this series, the assay was performed with 10 m&f c-u-ketoglutarate as the substrate. This substrate concentration is known to inhibit the soluble isoenzyme of ASAT to a high degree (17). During the period studied, the embryos do not feed at all. In subsequent experiments, the different enzymes responsible for the transamination of tyrosine were separated by isoelectric focusing as described earlier (17). Two fractions were consistently found at all of the developmental stages studied. One of them had a pI above 9 and was always connected with a peak of ASAT activity. This enzyme was slightly inhibited by 10 mM a-ketoglutarate. This peak is probably identical to mitochondrial aspartate aminotransferase, which can be liberated from mitochondria by detergents. At all stages studied, another fraction of tyrosine aminotransferase activity was found above pH 5. This fraction was strongly inhibited by 10 mM cY-ketoglutarate and it probably represents the cytoplasmic isoenzyme of ASAT. This peak is shown in Fig. la. From the age of 1 day on, another fraction was found that was not inhibited by (Yketoglutarate and had no aspartate aminotransferase activity (Fig. lb). This fraction probably represents specific tyrosine aminotransferase. This peak grew progressively larger during subsequent development. At stage 26, this fraction is already as tall as the fraction suggested to be identical to sASAT (see Fig. lb). At this stage, however, most of the total tyrosine aminotransferase activity seems to be due to the mitochondrial isoenzyme of ASAT. The exact time of appearance is difficult to assess, as marginal enzyme activities are to be measured. In Fig. 2, the electrofocusing of 2000 embryos at stage 18 (containing some stage 19, but no older embryos: age 19 hr f 15 min) is displayed. Here, three
428
DEVELOPMENTAL
0.:
BIOLOGY
0.1
J$&
b
56,
1977
cide with the rapid development of the foregut and the visceral pouches and arches (14). The percentages of total TAT activity due to each of the three enzymes capable of transaminating tyrosine at three different developmental stages are presented in Table 2. Tyrosine aminotransferase is known to be induced by tyrosine in the adult frog (16). To find a possible cause for the ap-
la
X f-L
* 0 c - 0.2 >
VOLUME
1 15
T 2(
15
50
Fu 4 0.1
0
FRACTION
FIG. 1. Electrofocusing of stages 1 (a) and 26 (b). The embryos were prepared as described in the text and focused in the pH range of 3.5-10. Only the beginning of the profile is shown: The peak in a corresponds to pH 5.6 and those in b correspond to pH values of 4.95 and 5.4. The enzyme activities are expressed directly as spectrophotometric readings. O-O, tyrosine aminotransferase activity with 1 mM a-ketoglutarate; O-O, tyrosine aminotransferase activity with 10 m&f cY-ketoglutarate; X-X , aspartate aminotransferase activity.
1 20
I
40
FRACTION
I
5’0
TABLE ARE
Stage 1 26 4’7/48
ACTIVITIES CAPABLE
18 34
18. About 2000 in the text and . = pH. The are as in Fig. 1.
2
OF THE THREE
OF TRANSAMINATING XenoDus EMBRYOS=
TAT (EC 2.6.1.5)
60
NUMBER
FIG. 2. Electrofocusing of stage embryos were prepared as described focused in the pH range of 4-6. other symbols and enzyme activities
RELATIVE
subforms of aspartate aminotransferase are seen, but specific tyrosine aminotransferase cannot be detected with certainty. The same held true for an experiment at stage 20, but, at stage 23, there are trace amounts of specific TAT. Shortly thereafter, at stage 26 (Fig. lb), this fraction is already prominent. At stage 47148, the “soluble” tyrosine transaminating activity is almost solely due to TAT. The appearance of specific tyrosine aminotransferase and the rapid growth of this fraction coin-
1
30
sASAT (EC 2.6.1.1) 9 11 3
ENZYMES
THAT
TYROSINE
IN
mASAT (EC 2.6.1.1) 91 71 63
n The relative activities are expressed as a percentage of the total activity. The quantitative relations were determined by electrofocusing the supernatant fractions of each stage, drawing the profile on paper, and cutting out the identified peaks, which were then weighed separately. a-Ketoglutarate was used at 1 m&f.
BRIEF STAGE ,o,,
1
10,
3s;73;
26,
8-
w z
lzi-
* 0 =4> I-
0
2
I
AGE
IN
3
4
DAYS
FIG. 3. The concentration of free tyrosine as a function of developmental stage. The embryos were prepared and the assays were performed as described in the text. Values are nanomoles per milligram of protein.
pearance of the enzyme in the embryo, the concentration of free tyrosine was estimated at different stages of development (Fig. 3). The initial concentration of this amino acid was very low, about one-sixth of the concentration in adult tissues. A steep increase begins at the age of about 20 hr, and a constant level (about 11 nmoles of tyrosinelmg of protein) is achieved on the fourth day of development. This rise in concentration closely precedes the appearance of specific tyrosine aminotransferase. It is known that the glycogen stores of the amphibian embryo are depleted during gastrulation (1). This has been proposed to be caused by hypoxia (2) or a cyclic AMPmediated control mechanism (7). It is possible that the rise in the concentration of free tyrosine, probably due to increased yolk breakdown, triggers the primary synthesis of tyrosine aminotransferase in the embryo. Previous depletion of the glycogen stores may play a permissive role. Here, glucocorticoids probably have no part, because the enzyme is not induced by them in the liver of the adult frog.
429
NOTES
Dopa decarboxylase (EC 4.1.1.26), the enzyme responsible for the synthesis of dopamine, is induced at the same time as tyrosine aminotransferase, while there seems to be no tyrosine decarboxylating activity during the early development of Xenopus (Huttunen, Ohisalo, and Pispa, submitted for publication). Thus, it seems that transamination, hydroxylation, and subsequent decarboxylation, leading to the synthesis of catecholamines or melanine, are the main routes of tyrosine metabolism, while direct decarboxylation and oxidation by monoamine oxidase seems to be insignificant. While this paper was in preparation, Fellman, Roth, and Fujita (5) reported on their studies, indicating that, in mammals, decarboxylation to tyramine is not a major route of tyrosine metabolism. This also seems to be the case with amphibians. The authors wish to thank Professor Sulo Toivonen for providing the frogs. The skillful technical assistance of Mrs. Alli Viljanen is gratefully acknowledged. This study was supported financially by the Sigrid Juselius Foundation. REFERENCES 1. BARBIERI, F. D., RAISMAN, J. S., and ALBARRACIN, C. (1967). Biol. Bull. 132, 299-310. 2. BARBIERI, F. D., and SALOMON, H. (1963). Acta
Embryol. Morphol. Exp. 6, 304-310. 3. CHAN, S., and COHEN, P. P. (1964). Arch. Bio&em. Biophys. 104, 335-337. 4. DAVID, J.-C., DAIRMAN, W., and UDENFRIEND, S. (1974). Arch. Biochem. Biophys. 160, 561568. 5. FELLMAN, J. H., ROTH, E. S., and FUJITA; T. S. (1976). Arch. Biochem. Biophys. 174, 562-567. 6. GREENGARD, O., and DEWEY, H. K. (1967). J. Biol. Chem. 242, 2986-2991. 7. GUSSECK, D. J., and HEDRICK, J. L. (1972). J. Biol. Chem. 247, 6603-6609. 8. HAGER, C. B., and KENNEY, F. T. (1968). J. Biol. Chem. 243, 3296-3300. 9. HOLTEN, D., and KENNEY, F. T. (1967). J. Biol. Chem. 242, 4372-4377. 10. IWASAKI, Y., LAMAR, C., DANENBERG, K., and PITOT, H. C. (1973). Eur. J. Biochem. 34, 347357. 11.
KOLER,
J.
H.,
R. D., VANBELLINGHEN, JONES, R. T., and
P. J., FELLMAN, BEHRMAN, R. E.
430
DEVELOPMENTAL
BIOLOGY
(1969). Science 163, 1348-1350. 12. LIN, E. C. C., and KNOX, W. E. (1958). J. Biol. Chem. 233, 1186-1189. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. I. (1951). J. Biol. Chem. 193, 265-275. 14. NIEUWKOOP, P. D., and FABER, J., eds. (1967). “Normal Tables of Xenopus laevis (Daudin).” North Holland, Amsterdam. 15. OHISALO, J. J., HASXNEN, I. E., and PISPA, J. P.
VOLUME
56, 1977
(1974). Biochim. Biophys. ACM 361, 48-52. 16. OHISALO, J. J., and PISPA, J. P. (1975). Biochim. Biophys. Actu 397, 94-100. 17. OHISALO, J. J., and PISPA, J. P. (1976). Acta Chem. Stand. B 30, 491-500. 18. R;41ti, N. C. R., and SCHWARTZ, A. L. (1973). Enzyme 15, 330-339. 19. UDENFRIEND, S., and COOPER, J. R. (1952). J. Biol. Chem. 196, 227-233. 20. WICKS, W. D. (1968). Science 160, 997-998.
BIOLOGY
Ontogeny
56, 426-430
of Tyrosine JORMA
Department
(1977)
Aminotransferase
J. OHISALO
of Medical
Chemistry, SF-001
Received
September
in Xenopus
AND JAAKKO
University 70 Helsinki 9,1976;
P. PISPA
of Helsinki, 17, Finland accepted
laevis
October
Siltavuorenpenger
10 A,
11,1976
The development of tyrosine aminotransferase (TAT) activity in Xenopus laevis embryos was studied. Undivided eggs can transaminate tyrosine to some extent. The enzyme activity increases after hatching on the third day of development. In the early stages of development, the transamination of tyrosine is due to aspartate aminotransferase (ASAT, EC 2.6.1.11, both isoenzymes of which are present in the undivided egg. No specific TAT (EC 2.6.1.5) can be detected until the age of about 1 day, at which time neurulation is complete and the rapid development of the foregut and visceral pouches and arches has begun. The appearance of the enzyme is immediately preceded by a steep increase in the concentration of free tyrosine. Tvrosine aminotransferase is known to be induced by its substrate in the adult liver, and a similar effect may operate in the embryo.
tyrosine aminotransferase during the embryonal development of the clawed toad, Xenopus laevis. Xenopus was chosen as the test animal because of the relative convenience of obtaining embryonal tissues in sufficient amounts and because the development of the embryo can be easily monitored visually.
INTRODUCTION
r.-Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5), TAT, is the enzyme mainly responsible for the catabolism of the amino acid tyrosine in the liver. In the rat the enzyme has very complicated hormonaI regulation (6, 8, 9, 12, 15, 20), while, in the frog, the enzyme activity has been shown to be affected only by tyrosine, dibutyryl cyclic AMP, and glucose (3, 16). The enzyme has several subforms, and the isoenzymes of aspartate aminotransferase (EC 2.6.1.1), ASAT, can be detected as “multiple forms” of tyrosine aminotransferase, due to their promiscuous substrate specificity (17). The mammalian enzyme is known to be inducible in the fetal liver (10) and in organ culture of fetal liver (13). The activity is increased at the time of birth and during the neonatal period in mammalian liver (11). The factors responsible for the increase are unknown, and the possibility that enzymes other than TAT might also transaminate tyrosine in vitro and hence cause false results has not been considered. The aim of the present work was to study the time of appearance of specific
MATERIALS
AND
METHODS
The frogs were a gift from Professor Sulo Toivonen from the Department of Zoology, University of Helsinki. They were housed, fed, and induced to lay eggs as advised by Nieuwkoop and Faber (14), whose normal tables were also used in staging. The staged embryos were counted and frozen immediately in liquid nitrogen. They were stored at -70°C. For assays, the embryos were allowed to thaw and were then homogenized in 3 vol of 0.5% Triton X-100. The homogenates were centrifuged at 10,OOOgfor 15 min, except for the electrofocusing experiments, for which they were centrifuged at 100,OOOgfor 60 min. After removing the fatty layer by aspiration, the supernatant fractions were used in the experiments. The isoelectric focusing was 426
Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
BRIEF
performed in a glycerol density gradient as described previously (17). The supernatant fractions from at least 500 embryos were divided equally between both gradient mixing vials. A pH gradient of 3.5-10 was generally used. The enzymes were assayed as described previously (17). Tyrosine was assayed by the method of Udenfriend and Cooper (19). The method of Lowry et al. (13) was used in protein estimations. The quantitative proportions of the different enzymes capable of transaminating tyrosine were estimated by drawing the isoelectric focusing profile on paper of even quality, cutting out the different peaks, and weighing them. The ampholines and the electrofocusing apparatus were obtained from LKB, Bromma, Sweden. The following reagents were used: L-tyrosine, pyridoxal-5’-phosphate (Sigma), ethylenediamine, glycerol, a-ketoglutaric acid (Merck), human chorionic gonadotropin (“Pregnyl,” Organon, Helsinki, Finland), and Triton X-100 (Koch-Light). The ASAT assay kit of Boehringer Mannheim, GmBH was used. RESULTS
AND
DISCUSSION
The development of total “tyrosine aminotransferase” activity is shown in Table 1. The initial activity is rather low, but an TABLE 1 TOTAL TYROSINE AMMINOTRANSFERASE ACTIVITY DURING THE EARLY DEVELOPMENT OF Xenopus laevis Stage
of development
TAT Picomoles of product per embryo per minute
1 18/19 23124 25126 27128 29/30 32 35/36 45146 47148
0.6 1.6 1.2 1.2 1.4 2.4 1.9 3.9 8.6 21.2
activity Picomoles of product per milligram of protein per minute 11 37 29 26 29 38 32 44 55 75
NOTES
427
increase is seen after stage 35136 i.e., at the age of 2.5 days. At this time, the walls of the primary hepatic cavity are folding up and starting to fill up the cavity. In this series, the assay was performed with 10 m&f c-u-ketoglutarate as the substrate. This substrate concentration is known to inhibit the soluble isoenzyme of ASAT to a high degree (17). During the period studied, the embryos do not feed at all. In subsequent experiments, the different enzymes responsible for the transamination of tyrosine were separated by isoelectric focusing as described earlier (17). Two fractions were consistently found at all of the developmental stages studied. One of them had a pI above 9 and was always connected with a peak of ASAT activity. This enzyme was slightly inhibited by 10 mM a-ketoglutarate. This peak is probably identical to mitochondrial aspartate aminotransferase, which can be liberated from mitochondria by detergents. At all stages studied, another fraction of tyrosine aminotransferase activity was found above pH 5. This fraction was strongly inhibited by 10 mM cY-ketoglutarate and it probably represents the cytoplasmic isoenzyme of ASAT. This peak is shown in Fig. la. From the age of 1 day on, another fraction was found that was not inhibited by (Yketoglutarate and had no aspartate aminotransferase activity (Fig. lb). This fraction probably represents specific tyrosine aminotransferase. This peak grew progressively larger during subsequent development. At stage 26, this fraction is already as tall as the fraction suggested to be identical to sASAT (see Fig. lb). At this stage, however, most of the total tyrosine aminotransferase activity seems to be due to the mitochondrial isoenzyme of ASAT. The exact time of appearance is difficult to assess, as marginal enzyme activities are to be measured. In Fig. 2, the electrofocusing of 2000 embryos at stage 18 (containing some stage 19, but no older embryos: age 19 hr f 15 min) is displayed. Here, three
428
DEVELOPMENTAL
0.:
BIOLOGY
0.1
J$&
b
56,
1977
cide with the rapid development of the foregut and the visceral pouches and arches (14). The percentages of total TAT activity due to each of the three enzymes capable of transaminating tyrosine at three different developmental stages are presented in Table 2. Tyrosine aminotransferase is known to be induced by tyrosine in the adult frog (16). To find a possible cause for the ap-
la
X f-L
* 0 c - 0.2 >
VOLUME
1 15
T 2(
15
50
Fu 4 0.1
0
FRACTION
FIG. 1. Electrofocusing of stages 1 (a) and 26 (b). The embryos were prepared as described in the text and focused in the pH range of 3.5-10. Only the beginning of the profile is shown: The peak in a corresponds to pH 5.6 and those in b correspond to pH values of 4.95 and 5.4. The enzyme activities are expressed directly as spectrophotometric readings. O-O, tyrosine aminotransferase activity with 1 mM a-ketoglutarate; O-O, tyrosine aminotransferase activity with 10 m&f cY-ketoglutarate; X-X , aspartate aminotransferase activity.
1 20
I
40
FRACTION
I
5’0
TABLE ARE
Stage 1 26 4’7/48
ACTIVITIES CAPABLE
18 34
18. About 2000 in the text and . = pH. The are as in Fig. 1.
2
OF THE THREE
OF TRANSAMINATING XenoDus EMBRYOS=
TAT (EC 2.6.1.5)
60
NUMBER
FIG. 2. Electrofocusing of stage embryos were prepared as described focused in the pH range of 4-6. other symbols and enzyme activities
RELATIVE
subforms of aspartate aminotransferase are seen, but specific tyrosine aminotransferase cannot be detected with certainty. The same held true for an experiment at stage 20, but, at stage 23, there are trace amounts of specific TAT. Shortly thereafter, at stage 26 (Fig. lb), this fraction is already prominent. At stage 47148, the “soluble” tyrosine transaminating activity is almost solely due to TAT. The appearance of specific tyrosine aminotransferase and the rapid growth of this fraction coin-
1
30
sASAT (EC 2.6.1.1) 9 11 3
ENZYMES
THAT
TYROSINE
IN
mASAT (EC 2.6.1.1) 91 71 63
n The relative activities are expressed as a percentage of the total activity. The quantitative relations were determined by electrofocusing the supernatant fractions of each stage, drawing the profile on paper, and cutting out the identified peaks, which were then weighed separately. a-Ketoglutarate was used at 1 m&f.
BRIEF STAGE ,o,,
1
10,
3s;73;
26,
8-
w z
lzi-
* 0 =4> I-
0
2
I
AGE
IN
3
4
DAYS
FIG. 3. The concentration of free tyrosine as a function of developmental stage. The embryos were prepared and the assays were performed as described in the text. Values are nanomoles per milligram of protein.
pearance of the enzyme in the embryo, the concentration of free tyrosine was estimated at different stages of development (Fig. 3). The initial concentration of this amino acid was very low, about one-sixth of the concentration in adult tissues. A steep increase begins at the age of about 20 hr, and a constant level (about 11 nmoles of tyrosinelmg of protein) is achieved on the fourth day of development. This rise in concentration closely precedes the appearance of specific tyrosine aminotransferase. It is known that the glycogen stores of the amphibian embryo are depleted during gastrulation (1). This has been proposed to be caused by hypoxia (2) or a cyclic AMPmediated control mechanism (7). It is possible that the rise in the concentration of free tyrosine, probably due to increased yolk breakdown, triggers the primary synthesis of tyrosine aminotransferase in the embryo. Previous depletion of the glycogen stores may play a permissive role. Here, glucocorticoids probably have no part, because the enzyme is not induced by them in the liver of the adult frog.
429
NOTES
Dopa decarboxylase (EC 4.1.1.26), the enzyme responsible for the synthesis of dopamine, is induced at the same time as tyrosine aminotransferase, while there seems to be no tyrosine decarboxylating activity during the early development of Xenopus (Huttunen, Ohisalo, and Pispa, submitted for publication). Thus, it seems that transamination, hydroxylation, and subsequent decarboxylation, leading to the synthesis of catecholamines or melanine, are the main routes of tyrosine metabolism, while direct decarboxylation and oxidation by monoamine oxidase seems to be insignificant. While this paper was in preparation, Fellman, Roth, and Fujita (5) reported on their studies, indicating that, in mammals, decarboxylation to tyramine is not a major route of tyrosine metabolism. This also seems to be the case with amphibians. The authors wish to thank Professor Sulo Toivonen for providing the frogs. The skillful technical assistance of Mrs. Alli Viljanen is gratefully acknowledged. This study was supported financially by the Sigrid Juselius Foundation. REFERENCES 1. BARBIERI, F. D., RAISMAN, J. S., and ALBARRACIN, C. (1967). Biol. Bull. 132, 299-310. 2. BARBIERI, F. D., and SALOMON, H. (1963). Acta
Embryol. Morphol. Exp. 6, 304-310. 3. CHAN, S., and COHEN, P. P. (1964). Arch. Bio&em. Biophys. 104, 335-337. 4. DAVID, J.-C., DAIRMAN, W., and UDENFRIEND, S. (1974). Arch. Biochem. Biophys. 160, 561568. 5. FELLMAN, J. H., ROTH, E. S., and FUJITA; T. S. (1976). Arch. Biochem. Biophys. 174, 562-567. 6. GREENGARD, O., and DEWEY, H. K. (1967). J. Biol. Chem. 242, 2986-2991. 7. GUSSECK, D. J., and HEDRICK, J. L. (1972). J. Biol. Chem. 247, 6603-6609. 8. HAGER, C. B., and KENNEY, F. T. (1968). J. Biol. Chem. 243, 3296-3300. 9. HOLTEN, D., and KENNEY, F. T. (1967). J. Biol. Chem. 242, 4372-4377. 10. IWASAKI, Y., LAMAR, C., DANENBERG, K., and PITOT, H. C. (1973). Eur. J. Biochem. 34, 347357. 11.
KOLER,
J.
H.,
R. D., VANBELLINGHEN, JONES, R. T., and
P. J., FELLMAN, BEHRMAN, R. E.
430
DEVELOPMENTAL
BIOLOGY
(1969). Science 163, 1348-1350. 12. LIN, E. C. C., and KNOX, W. E. (1958). J. Biol. Chem. 233, 1186-1189. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. I. (1951). J. Biol. Chem. 193, 265-275. 14. NIEUWKOOP, P. D., and FABER, J., eds. (1967). “Normal Tables of Xenopus laevis (Daudin).” North Holland, Amsterdam. 15. OHISALO, J. J., HASXNEN, I. E., and PISPA, J. P.
VOLUME
56, 1977
(1974). Biochim. Biophys. ACM 361, 48-52. 16. OHISALO, J. J., and PISPA, J. P. (1975). Biochim. Biophys. Actu 397, 94-100. 17. OHISALO, J. J., and PISPA, J. P. (1976). Acta Chem. Stand. B 30, 491-500. 18. R;41ti, N. C. R., and SCHWARTZ, A. L. (1973). Enzyme 15, 330-339. 19. UDENFRIEND, S., and COOPER, J. R. (1952). J. Biol. Chem. 196, 227-233. 20. WICKS, W. D. (1968). Science 160, 997-998.
