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Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis
Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis D. L. HUBBARD and R. W. ATHERTON Department of Zoology-Physiology The University of Wyoming Laramie. Wyoming 82071, U.S.A.
Received October 1974fRevised March 1975
Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.
Introduction
Anuran metamorphosis involves the loss of tadpole tail $issuewith the simultaneous growth and differentiation of the adult organism. Proteins are the major cell constituent disposed of during tail regression [ 1I and the mechanisms involved in the loss of protein from tail tissue appear to be at least two-fold. First, there is an alteration in the normal balance between protein synthesis and protein degradation prior to morphological signs of resorption [ll. The second mechanism involves the appearance of macrophages and increased hydrolase activity [2]. During metamorphosis increased activity of many proteolytic enzymes has been reported [2,31. The requirement for RNA synthesis during tail resorption was first shown by the delay of tail regression and supression of lysosomal enzyme activity in response to actinomycin-D injection [2,41. In vitro tail-tip regression induced by 3,5,3’-Triiodothyronine (T3)was inhibited by protein synthesis blocking agents cycloheximide,puromycin 151, and by RNA synthesis blocking actinomycin D, thus showing the requirement for protein and RNA synthesis during in vitro tail-tip regression [51. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis has been developed and shown to give molecular-weight estimation of polypeptide chains [61. The reliability of polypeptide molecular-weight determination by this technique has been shown to be accurate within 10% of peptide weight 171. Differentiation 4, 153-158 (1975)
-
0 by Springer-Verlag 1975
The purpose of this study was to classify the major subunit proteins of Xenopus laevis tail tissue according to molecular weight during selected phases of development during in vivo metamorphosis.
Methods Materials. The proteins and chemicals and their sources were as follows: bovine serum albumin, bovine erythrocyte carbonic anhydrase, bovine liver catalase, egg white lysozyme, sodium dodecyl sulfate, Coomassie Brilliant Blue R (Sigma Chemical Company); E . coli /3 galactosidase (Worthington Biochemical Corporation); rabbit muscle creatine kinase (Boehringer Mannheim); riboflavin, N.N’-methylenebisacrylamide (Eastman Kodak Company); ammonium persulfate, N.N,N’,N’-tetramethylethylenediamine(Canal Industry Corporation); tris (hydroxy-methyl) aminomethane, sucrose (enzyme grade) (SchwardMann); glycine, 2-mercaptoethanol, Cleland’s Reagent (dithiothreitol) (Calbiochem Chemical Company); acrylamide (B D H Chemicals Ltd.); dialysis tubing (Union Carbide, Food Products Division); sodium carbonate and potassium phosphate monobasic (Mallinckrodt Chemicals), human chorionic gonadotrophin, (Nutritional Biochemicals), and sodium phosphate dibasic heptahydrate (J. T. Baker Chemical Company). Methods. Male and female Xenopus laevis were induced into mating behaviour and subsequent production of zygotes by a series of injections of human chorionic gonadotropin hormone. Embryos were staged according to external and internal features. All embryos were raised in a controlled 12-h light cycle on a diet of spinach. As embryos approached metamorphosis they were transferred to shallow pans and fed liver. Animals were generally caredfor in such amanner as to insure
154
D. L. Hubbard and R. W. Atherton: Gels were pre-electrophoresed at 2 ma/tube for 45 min with reversed polarity followed by 15 min with normal polarity. Between 5 and 50 pl of the protein sample were layered on the gels. Electrophoresis was performed at 4 ma/tube in a water-jacketed reservoir at 1loC. Gelswereelectrophoresedfor45 min'oruntilthebromophenol blue tracker was 0.5 cm from the bottom of the gel column. Staining for 5-8 h and destaining for 8-12 h was done 171. The absolute quantitation of proteins by the intensity of Coomassie Blue staining is not possible without the availability of identical reference proteins [141. All destained gels were scanned at 570 nm in a Giford 240 spectrophotometer equipped with a model 24 10 linear transport at a scanning speed of 1 cm/min. Analysis of the bands was accomplished using the gel scans as presented in Figs. 3-7. R, values were calculated according to a normalising equation [71. Molecular weights were determined from a plot of -100 log R,versus molecular weight (Fig. 2) as previously described [151.
metamorphosis was complete and were ascertained as normal by staging and observations of normal rates of metamorphosis. The stages of embryos used in this study ranged from premetamorphic stage 45 through the following stages of metamorphosis; 52-53, 57-58, 61-62, and 63-64 191. Tails were excisedjust posterior to the anal pore, rinsed three times with sterile phosphate buffer, weighed, diced, and homogenised in a pH 8 phosphate buffer (0.1 g wet tissue/ml buffer) using a glass mortar and a motor-driven Teflon pestle. Homogenates consisted of tissue pooled from 10 to 70 embryos of each stage. The supernatant was then collected and analysed for total protein content [lo]. The supernatant was stored at4O C for 3-6 h before preparation for electrophoresis. The pH 8.0phosphate buffer employed consisted of 94.5% of M/15 Na,HPO,. 12 H,O and 5.5% of M/15 KH,PO, [ I l l . The concentration of protein I101 in the supernatant was usually in the desired range for electrophoresis; however, when necessary the supernatant was diluted to the desired concentration (2.0 f 0.03 mg/ml) using pH 8.0 phosphate buffer. To some volume of supernatant ofdesired protein concentration,usuaIly 1 ml, 8 mg of SDS per mg of protein was added, and samples were mixed for one minute. Next 10% (v/v) 2-mercaptoethanol was added to samples which were then incubated at room temperature for 8-12 h. After incubation, samples were dialysed for 5-8 h against the electrophoresis buffer containing 0.1% SDS, 0.5% dithiothreitol, 2% enzyme grade sucrose, and 0.005% bromophenol blue. The crystalline molecular-weight marker proteins were dissolved in 0.05 M Na,CO, to a concentration of 1.0 f 0.03 mg/ml. The molecular weight marker proteins were prepared, incubated, and dialysed as discussed above. A 5 cmcolumnof a 7.5% polyacrylamide separating gel with 1 cm 2.5% polyacrylamide stacking gel was prepared 112, 131. Theelectrophoresis buffer was 0.5 M t r i s (hydroxy-methyl) amino-methane and 0.38 M glycine adjusted to pH 8.4. In addition, the upper reservoir buffer contained 0.1% SDS. Other techniques were explored, which incorporated longer gels and different buffers. However, the most consistent results were obtained with a 5 cm gel and a tris-glycine buffer.
Results
Molecular weight calibration of gels is shown in Fig. 1. Lysozyme migrated with and masked the bromophenol blue tracker dye on stained gels (Fig. 1G). The calculated -100 log R,values are reportedin Table I and graphically presented in Fig. 2. Between 20 and 24 protein subunits ranging in molecular weight from 200,000 to 15,000 daltons were resolved from the tail tissue of each stage studied (Figs. 3-7). Molecular weight classification of the subunit proteins is presented in Table 2. The molecular weights of all subunits are significantly different (p5 0.05) by the student t-test. A total of nineteen subunit proteins was resolved from the tail tissue of stage45 tadpoles. An increase in the
BPB+
A
B
C
D
E
F
G
Fig. 1. Coomassie blue stained 7.5% polyacrylamide gels prepared to show the relative migration of proteins of known molecular weights. Arrows indicate major subunit proteins. RPB = bromophenol h1ue.A P-galactosidase; B Phosphorylasea; CBovine serum albumin; D Catalase; E Creatine kinase; F Carbonic anhydrase; G Lysozyme
155
Protein Changes during Metamorphosis
Table 1. Relative migration of molecular weight marker proteins, mean R, values, and -100 log Rp Protein standard
Subunit M.W. Ref (#)
P-galactosidase Phosphorylase a Bovine serum albumin Catalase Creatine kinase Carbonic anhydrase Lysozyme
130,000 92,500 68,000 57,000 40,000 29,000 14,300
(15, 16) (17) (15, 7) (18, 19) (7) (7) (7)
Corrected mean R, S.E. (n)
-100 log Mean R,
0.2149 & 0.2501 f 0.4033 k 0.4729 & 0.6374 f 0.7682 k 0.9658 k
66.7731 60.1839 39.4328 32.5262 19.5602 11.4508 1.5100
.0021 .0029 .0031 .0043
(2) (2) (9) (3) .0045 (7) .0085 (6) .005 (2)
7. Weber and Osborn, 1969. 16. Ullmann et al., 1968. 17. Waugh, 1954. 18. Madsen and Cori, 1956. 19. Schroeder et al., 1969. 20. Tanford and Lovrien. 1962. 21. Dawson et al., 1967.
Table 2. Band patterns and average subunit molecular weight for stages 45 through 64 of Xenopus laevis development.
Band #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
22 23 24
(Average M.W. S.E.) x 10-3
197.0 155.5 140.0 136.0 125.0 114.0 102.0 92.0 84.0 77.0 72.0 68.0 59.0 57.0 53.0 47.0 37.0 33.0 28.0 25.0 23.0 19.0 16.5 15.0
2.44 1.10 1.20 1.32 0.67 0.77 0.38 0.5 1 0.35 0.74
*
1.03 0.43
*
-
Band absent
52-53
57-58
+ + + + + + + + +
~-
-
-
-
+ +
+ +
+ +
+ +
+
+ + + + + +
*
+
+
*
value
64
+
+
+ +
+ + +
+ + + + +
+
+
-
-
0.50 0.13
61-62
+ + + + + + + + + +
0.3 1 0.4 I 0.40 0.39 0.61 0.31
* S.E. Determined for average R,
+ Band present
Stage 45
+ +
t
+ + + +
-
-
+ +
+ +
-
+
+ -
+
156
D. L. Hubbard and R. W. Atherton:
80-
-
60-
-
40-
-
0
A
2
0
!? I
Fig. 2. The negative logarithm of relative mobility (Rc) versus subunit molecular weight of the marker proteins, r = 0.9784. (see also Fig. 1 and Table 1). Conditions for electrophoresis of marker proteins is presented in the Materials and Methods. A P-galactosidase; B Phosphorylase a; CBovine serum albumin;D Cata1ase;ECreatine kinase;F Carbonic anhydrase; G Lysozyme
20- -
20
40
do
80
I20
100
MOLECULAR W l l G H l x 10-l
m
a n
f
I 1-1
(+I
Fig. 3. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stage 45 of Xenopus luevis development (Band numbers, BPB = Bromophenol blue)
(-1
(+)
Fig. 4. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stages 52-53 of Xenopus laevis development (Band numbers, BPB = Bromophenol blue)
157
Protein Changes during Metamorphosis
number of subunit proteins resolved from stage 52-53 (Fig. 4) tail tissue was observed by resolution of a 25,000 dalton subunit not seenin stage 45 (Fig. 3). The 20 other subunit proteins isolated from stage 52-53 (Fig. 4) have the same average molecular weight as the subunit proteins of stage 45. Twenty-one subunit proteins were observed in tissue of stages 57-58 (Fig. 5 ) and 61-62 (Fig. 6) with the same average molecular weight as reported for stage 52-53 (Fig. 4). Three protein subunits (molecular weights 72,000,23,000 and 15,OOO)previously not resolved for any other ages were seen in the homogenates of stage 64 (Fig. 7) with the remaining 21 protein subunits having the same average molecular weights as reported for other stages.
Discussion
Stage 45 tadpoles have no morphological signs of metamorphosis 191. Thus, the protein subunit peptides isolated from prepared samples of tail tissue of this age should represent the base population of proteins subunits present in Xenopus laevis tadpole tail tissue. An overall decrease in the rate of protein synthesis occurs in the tail during metamorphosis [ll; although evidence exists showing an increase in the synthesis of specific proteins probably resulting from macrophage activation [21. An increase in the activity of severalproteolytic enzymes has been documented and is probably the result of a directed stimulation ofprotein synthesis 131.Our
!
t
1
E
0
s;
0 0
(-1
I +)
Fig. 5. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stages 57-58 of Xenopus laevis development (Band numbers, BPB = Bromophenol blue)
I-)
(+I
Fig. 6. SDS polyacrylamide gel electrophoresis of tail-tissue proteins
from stages 61-62 of Xenopus Zaevis development (Band numbers, BPB = Bromophenol blue)
D. L. Hubbard and R. W. Atherton
158 study shows an increase in the number of subunits resolved in stage 52-53 embryos (Fig. 4) when compared to stage 45 (Fig. 3). Thenew 25,000 subunit may represent a new gene product or may be indicative of increased protein synthesis of a proteolytic enzyme subunit protein. The number and molecular weight of subunit proteins remain constant during stages 52-53,57-58, and 6 1-62 (Figs. 4-6, respectively) ofmetamorphosis where no morphological signs of tail resorption occurs. An increase in the rate ofprotein degradation occurs at the time of weight and length loss during tail resorption [ll. It is during this period, stage 64 (Fig. 7) that our results show 3 new protein subunits which may be degradation products of larger protein subunits. Alteration in the balance between protein synthesis and degradation appears to have little effect on the subunit
protein population of amphibian tail tissue prior to morphological signs of tail regression. Whether studies with metabolic blocking agents will show an altered subunit protein profile has yet to be determined but is currently being investigated in our laboratory. New protein subunits observed in this study are probably indicative of proteolytic enzyme production prior to the phenomenon of tail regression. In conclusion, during the period of tail regression, the subunit protein population shows signs of degradation as indicated by an increase in the number of protein subunits. Acknowledgments: The authors wish to express their appreciation for financial support from the University of Wyoming Division of Basic Research and the University of Wyoming Graduate School and Federal Minority Aid Program. A special note of appreciation to theUniversityofWyomingSchoolofAgriculture-Department ofBiochemistry.
Dedicated to the memory of Professor Gordon M. Ramm 1921-1975.
References E.: Anal. Bio. Chem. 20, 150, 1967 2. Weber, R.: Biochemistry of Animal Development, Vol. 11, p. 227. New York: Academic Press 1967 3. Frieden, E., Jus, J . J.: Biochemical Actions of Hormones, Vol. I, Litwach (ed.), p. 1. New York: Academic Press. 1970 4. Tonoue, T., Frieden, E.: J. Biol. Chem. 245, 2359, 1970 5. Tata, J . R. Deu. B i d . 13, 77. 1966 6. Shapiro, A. L., Vinuela, E., Maize], J. V.: Biochem. Biophys. Res. Comun. 28, 815, 1967 7. Weber, K., Osborn, M.: J. Biol. Chem. 244, 4406, 1969 8. deleted 9. Nieuw Koop, P. D., Faher, J. (eds.): Normal Table of Xenopus laeuis (Dauden). A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis. 243 pp. Amsterdam: North-Holland Publishing Co. 1956 10. Lowry, 0. H., Rosenbrough. N. J., Farr, A. L., Randall, R. J.: J. Biol. Chem. 193, 265, 1951 11. Ellman, F. L., Coutiney, K. D., Andres, V., Featherstone, R. M.: Biochem. Pharmacol. I , 88, 1961 12. Ornstein, L.: Ann. N.Y. Acad. Sci. 121, 321, 1964 13. Davis, B. J.: Ann. N.Y. Acad. Sci. 121, 404, 1964 14. Chrambach,A., Reisfeld, A., Wyckoff,M., Zaccari, J.:Anal. Bio. Chem. 20, 150, 1967 15. Neville, D. M.: J. Biol. Chem. 246, 6328, 1971 16. Ullmann, A,, Goldberg, M. E., Perrin, D., Monod: J. Biochem. 1, 261, 1968 17. Waugh, I).F.: Adv. Prot. Chem. 9, 325, 1954 18. Madsen, M. B., Cori, C. F.: J. Biol. Chern. 223, 1055, 1956 19. Schroeder, W. A., Shelton, J. R., Shelton, J. B., Robberson, B., Apell, G.: Arch. Biochem. Biophys. 131, 653, 1969 20. Tanford, C., Lovrien, R.: J. Amer. Chem. Soc. 84, 1892, 1962 21. Dawson, D. M., Eppenberger, H. S., Kaplan, N. O.:J.Biol.Chem. 242, 211, 1967 1. Little, G. H., Atkinson, B. G., Frieden,
\
1-1
(+I
Fig. 7. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stage 64 of Xenopus laeuis development (Band numbers, BPB = Bromophenol blue)
Received October 1974fRevised March 1975
Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.
Introduction
Anuran metamorphosis involves the loss of tadpole tail $issuewith the simultaneous growth and differentiation of the adult organism. Proteins are the major cell constituent disposed of during tail regression [ 1I and the mechanisms involved in the loss of protein from tail tissue appear to be at least two-fold. First, there is an alteration in the normal balance between protein synthesis and protein degradation prior to morphological signs of resorption [ll. The second mechanism involves the appearance of macrophages and increased hydrolase activity [2]. During metamorphosis increased activity of many proteolytic enzymes has been reported [2,31. The requirement for RNA synthesis during tail resorption was first shown by the delay of tail regression and supression of lysosomal enzyme activity in response to actinomycin-D injection [2,41. In vitro tail-tip regression induced by 3,5,3’-Triiodothyronine (T3)was inhibited by protein synthesis blocking agents cycloheximide,puromycin 151, and by RNA synthesis blocking actinomycin D, thus showing the requirement for protein and RNA synthesis during in vitro tail-tip regression [51. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis has been developed and shown to give molecular-weight estimation of polypeptide chains [61. The reliability of polypeptide molecular-weight determination by this technique has been shown to be accurate within 10% of peptide weight 171. Differentiation 4, 153-158 (1975)
-
0 by Springer-Verlag 1975
The purpose of this study was to classify the major subunit proteins of Xenopus laevis tail tissue according to molecular weight during selected phases of development during in vivo metamorphosis.
Methods Materials. The proteins and chemicals and their sources were as follows: bovine serum albumin, bovine erythrocyte carbonic anhydrase, bovine liver catalase, egg white lysozyme, sodium dodecyl sulfate, Coomassie Brilliant Blue R (Sigma Chemical Company); E . coli /3 galactosidase (Worthington Biochemical Corporation); rabbit muscle creatine kinase (Boehringer Mannheim); riboflavin, N.N’-methylenebisacrylamide (Eastman Kodak Company); ammonium persulfate, N.N,N’,N’-tetramethylethylenediamine(Canal Industry Corporation); tris (hydroxy-methyl) aminomethane, sucrose (enzyme grade) (SchwardMann); glycine, 2-mercaptoethanol, Cleland’s Reagent (dithiothreitol) (Calbiochem Chemical Company); acrylamide (B D H Chemicals Ltd.); dialysis tubing (Union Carbide, Food Products Division); sodium carbonate and potassium phosphate monobasic (Mallinckrodt Chemicals), human chorionic gonadotrophin, (Nutritional Biochemicals), and sodium phosphate dibasic heptahydrate (J. T. Baker Chemical Company). Methods. Male and female Xenopus laevis were induced into mating behaviour and subsequent production of zygotes by a series of injections of human chorionic gonadotropin hormone. Embryos were staged according to external and internal features. All embryos were raised in a controlled 12-h light cycle on a diet of spinach. As embryos approached metamorphosis they were transferred to shallow pans and fed liver. Animals were generally caredfor in such amanner as to insure
154
D. L. Hubbard and R. W. Atherton: Gels were pre-electrophoresed at 2 ma/tube for 45 min with reversed polarity followed by 15 min with normal polarity. Between 5 and 50 pl of the protein sample were layered on the gels. Electrophoresis was performed at 4 ma/tube in a water-jacketed reservoir at 1loC. Gelswereelectrophoresedfor45 min'oruntilthebromophenol blue tracker was 0.5 cm from the bottom of the gel column. Staining for 5-8 h and destaining for 8-12 h was done 171. The absolute quantitation of proteins by the intensity of Coomassie Blue staining is not possible without the availability of identical reference proteins [141. All destained gels were scanned at 570 nm in a Giford 240 spectrophotometer equipped with a model 24 10 linear transport at a scanning speed of 1 cm/min. Analysis of the bands was accomplished using the gel scans as presented in Figs. 3-7. R, values were calculated according to a normalising equation [71. Molecular weights were determined from a plot of -100 log R,versus molecular weight (Fig. 2) as previously described [151.
metamorphosis was complete and were ascertained as normal by staging and observations of normal rates of metamorphosis. The stages of embryos used in this study ranged from premetamorphic stage 45 through the following stages of metamorphosis; 52-53, 57-58, 61-62, and 63-64 191. Tails were excisedjust posterior to the anal pore, rinsed three times with sterile phosphate buffer, weighed, diced, and homogenised in a pH 8 phosphate buffer (0.1 g wet tissue/ml buffer) using a glass mortar and a motor-driven Teflon pestle. Homogenates consisted of tissue pooled from 10 to 70 embryos of each stage. The supernatant was then collected and analysed for total protein content [lo]. The supernatant was stored at4O C for 3-6 h before preparation for electrophoresis. The pH 8.0phosphate buffer employed consisted of 94.5% of M/15 Na,HPO,. 12 H,O and 5.5% of M/15 KH,PO, [ I l l . The concentration of protein I101 in the supernatant was usually in the desired range for electrophoresis; however, when necessary the supernatant was diluted to the desired concentration (2.0 f 0.03 mg/ml) using pH 8.0 phosphate buffer. To some volume of supernatant ofdesired protein concentration,usuaIly 1 ml, 8 mg of SDS per mg of protein was added, and samples were mixed for one minute. Next 10% (v/v) 2-mercaptoethanol was added to samples which were then incubated at room temperature for 8-12 h. After incubation, samples were dialysed for 5-8 h against the electrophoresis buffer containing 0.1% SDS, 0.5% dithiothreitol, 2% enzyme grade sucrose, and 0.005% bromophenol blue. The crystalline molecular-weight marker proteins were dissolved in 0.05 M Na,CO, to a concentration of 1.0 f 0.03 mg/ml. The molecular weight marker proteins were prepared, incubated, and dialysed as discussed above. A 5 cmcolumnof a 7.5% polyacrylamide separating gel with 1 cm 2.5% polyacrylamide stacking gel was prepared 112, 131. Theelectrophoresis buffer was 0.5 M t r i s (hydroxy-methyl) amino-methane and 0.38 M glycine adjusted to pH 8.4. In addition, the upper reservoir buffer contained 0.1% SDS. Other techniques were explored, which incorporated longer gels and different buffers. However, the most consistent results were obtained with a 5 cm gel and a tris-glycine buffer.
Results
Molecular weight calibration of gels is shown in Fig. 1. Lysozyme migrated with and masked the bromophenol blue tracker dye on stained gels (Fig. 1G). The calculated -100 log R,values are reportedin Table I and graphically presented in Fig. 2. Between 20 and 24 protein subunits ranging in molecular weight from 200,000 to 15,000 daltons were resolved from the tail tissue of each stage studied (Figs. 3-7). Molecular weight classification of the subunit proteins is presented in Table 2. The molecular weights of all subunits are significantly different (p5 0.05) by the student t-test. A total of nineteen subunit proteins was resolved from the tail tissue of stage45 tadpoles. An increase in the
BPB+
A
B
C
D
E
F
G
Fig. 1. Coomassie blue stained 7.5% polyacrylamide gels prepared to show the relative migration of proteins of known molecular weights. Arrows indicate major subunit proteins. RPB = bromophenol h1ue.A P-galactosidase; B Phosphorylasea; CBovine serum albumin; D Catalase; E Creatine kinase; F Carbonic anhydrase; G Lysozyme
155
Protein Changes during Metamorphosis
Table 1. Relative migration of molecular weight marker proteins, mean R, values, and -100 log Rp Protein standard
Subunit M.W. Ref (#)
P-galactosidase Phosphorylase a Bovine serum albumin Catalase Creatine kinase Carbonic anhydrase Lysozyme
130,000 92,500 68,000 57,000 40,000 29,000 14,300
(15, 16) (17) (15, 7) (18, 19) (7) (7) (7)
Corrected mean R, S.E. (n)
-100 log Mean R,
0.2149 & 0.2501 f 0.4033 k 0.4729 & 0.6374 f 0.7682 k 0.9658 k
66.7731 60.1839 39.4328 32.5262 19.5602 11.4508 1.5100
.0021 .0029 .0031 .0043
(2) (2) (9) (3) .0045 (7) .0085 (6) .005 (2)
7. Weber and Osborn, 1969. 16. Ullmann et al., 1968. 17. Waugh, 1954. 18. Madsen and Cori, 1956. 19. Schroeder et al., 1969. 20. Tanford and Lovrien. 1962. 21. Dawson et al., 1967.
Table 2. Band patterns and average subunit molecular weight for stages 45 through 64 of Xenopus laevis development.
Band #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
22 23 24
(Average M.W. S.E.) x 10-3
197.0 155.5 140.0 136.0 125.0 114.0 102.0 92.0 84.0 77.0 72.0 68.0 59.0 57.0 53.0 47.0 37.0 33.0 28.0 25.0 23.0 19.0 16.5 15.0
2.44 1.10 1.20 1.32 0.67 0.77 0.38 0.5 1 0.35 0.74
*
1.03 0.43
*
-
Band absent
52-53
57-58
+ + + + + + + + +
~-
-
-
-
+ +
+ +
+ +
+ +
+
+ + + + + +
*
+
+
*
value
64
+
+
+ +
+ + +
+ + + + +
+
+
-
-
0.50 0.13
61-62
+ + + + + + + + + +
0.3 1 0.4 I 0.40 0.39 0.61 0.31
* S.E. Determined for average R,
+ Band present
Stage 45
+ +
t
+ + + +
-
-
+ +
+ +
-
+
+ -
+
156
D. L. Hubbard and R. W. Atherton:
80-
-
60-
-
40-
-
0
A
2
0
!? I
Fig. 2. The negative logarithm of relative mobility (Rc) versus subunit molecular weight of the marker proteins, r = 0.9784. (see also Fig. 1 and Table 1). Conditions for electrophoresis of marker proteins is presented in the Materials and Methods. A P-galactosidase; B Phosphorylase a; CBovine serum albumin;D Cata1ase;ECreatine kinase;F Carbonic anhydrase; G Lysozyme
20- -
20
40
do
80
I20
100
MOLECULAR W l l G H l x 10-l
m
a n
f
I 1-1
(+I
Fig. 3. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stage 45 of Xenopus luevis development (Band numbers, BPB = Bromophenol blue)
(-1
(+)
Fig. 4. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stages 52-53 of Xenopus laevis development (Band numbers, BPB = Bromophenol blue)
157
Protein Changes during Metamorphosis
number of subunit proteins resolved from stage 52-53 (Fig. 4) tail tissue was observed by resolution of a 25,000 dalton subunit not seenin stage 45 (Fig. 3). The 20 other subunit proteins isolated from stage 52-53 (Fig. 4) have the same average molecular weight as the subunit proteins of stage 45. Twenty-one subunit proteins were observed in tissue of stages 57-58 (Fig. 5 ) and 61-62 (Fig. 6) with the same average molecular weight as reported for stage 52-53 (Fig. 4). Three protein subunits (molecular weights 72,000,23,000 and 15,OOO)previously not resolved for any other ages were seen in the homogenates of stage 64 (Fig. 7) with the remaining 21 protein subunits having the same average molecular weights as reported for other stages.
Discussion
Stage 45 tadpoles have no morphological signs of metamorphosis 191. Thus, the protein subunit peptides isolated from prepared samples of tail tissue of this age should represent the base population of proteins subunits present in Xenopus laevis tadpole tail tissue. An overall decrease in the rate of protein synthesis occurs in the tail during metamorphosis [ll; although evidence exists showing an increase in the synthesis of specific proteins probably resulting from macrophage activation [21. An increase in the activity of severalproteolytic enzymes has been documented and is probably the result of a directed stimulation ofprotein synthesis 131.Our
!
t
1
E
0
s;
0 0
(-1
I +)
Fig. 5. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stages 57-58 of Xenopus laevis development (Band numbers, BPB = Bromophenol blue)
I-)
(+I
Fig. 6. SDS polyacrylamide gel electrophoresis of tail-tissue proteins
from stages 61-62 of Xenopus Zaevis development (Band numbers, BPB = Bromophenol blue)
D. L. Hubbard and R. W. Atherton
158 study shows an increase in the number of subunits resolved in stage 52-53 embryos (Fig. 4) when compared to stage 45 (Fig. 3). Thenew 25,000 subunit may represent a new gene product or may be indicative of increased protein synthesis of a proteolytic enzyme subunit protein. The number and molecular weight of subunit proteins remain constant during stages 52-53,57-58, and 6 1-62 (Figs. 4-6, respectively) ofmetamorphosis where no morphological signs of tail resorption occurs. An increase in the rate ofprotein degradation occurs at the time of weight and length loss during tail resorption [ll. It is during this period, stage 64 (Fig. 7) that our results show 3 new protein subunits which may be degradation products of larger protein subunits. Alteration in the balance between protein synthesis and degradation appears to have little effect on the subunit
protein population of amphibian tail tissue prior to morphological signs of tail regression. Whether studies with metabolic blocking agents will show an altered subunit protein profile has yet to be determined but is currently being investigated in our laboratory. New protein subunits observed in this study are probably indicative of proteolytic enzyme production prior to the phenomenon of tail regression. In conclusion, during the period of tail regression, the subunit protein population shows signs of degradation as indicated by an increase in the number of protein subunits. Acknowledgments: The authors wish to express their appreciation for financial support from the University of Wyoming Division of Basic Research and the University of Wyoming Graduate School and Federal Minority Aid Program. A special note of appreciation to theUniversityofWyomingSchoolofAgriculture-Department ofBiochemistry.
Dedicated to the memory of Professor Gordon M. Ramm 1921-1975.
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Fig. 7. SDS polyacrylamide gel electrophoresis of tail-tissue proteins from stage 64 of Xenopus laeuis development (Band numbers, BPB = Bromophenol blue)