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Acid Phosphatase in the Tail of Xenopus laevis During Development and Metamorphosis '

HELEN ROBINSON Department of Biology, Yale University

ABSTRACT Quantitative and qualitative changes in acid phosphatase during growth and metamorphosis of the tail of Xenopus laevis have been investigated. Enzyme activity was assayed by two methods using different substrates, p-nitrophenyl phosphate and a-glycerophosphate, The enzyme was found to be consistently more active with p-nitrophenyl phosphate. Specific activity of whole tail homogenates assayed by both methods remains constant during development and early metamorphosis, and then rises sharply as the rate of tail regression begins to accelerate. Total phosphatase activity of whole tail homogenates rises linearly during development and early metamorphosis, and then maintains a high level during the first half of the period of tail regression. After this it declines. Regional localization of acid phosphatase activity has been determined in different parts of the tail: dorsal fin, ventral fin, and muscle. Changes in the apparent Km and pH optimum of acid phosphatase occur during tail regression. These properties remain constant during development and early metamorphosis, and are then found to change approximately in parallel with changes in enzyme activity during tail resorption. Possible implications of these biochemical events are discussed.

The enzymatic changes associated with tail growth and regression in anurans of- fer an unusually favorable system for study of the biochemical aspects of cell differen- tiation and cell death as influenced by hormone action. Biochemical studies on tail tissues during metamorphosis have been concentrated on quantitative variations in selected enzymes known to have catabolic significance, in particular collagenase and the acid hydrolases characteristic of lysosomes. Studies on collagenase (Lapiere and Gross, '63), cathepsin (Weber, '57a,b, '63; Eeckhout, '65), acid phosphatase (Weber, '61, '63, '64; Eeckhout, '65), p-glucuronidase (Price and Frieden, '63; Kubler and Frieden, '64; Eeckhout, '65), ,&-galactosi- dase (Eeckhout, '65), acid DNase (Cole- man, '62, '63), and acid RNase (Eeckhout, '65), all indicate that these enzymes increase in activity during tail atrophy. The work of Tata ('66) has shown that protein and RNA synthesis are stimulated during tail regression and that regression is blocked by inhibition of these processes (Weber, '65; Tata, '66). The data which have been accumulated strongly support the hypothesis that tail atrophy is accom- plished by means of hydrolytic enzymes which are synthesized de nouo just prior to or during the onset of metamorphosis

J. EXP. ZOOL.. 173: 215-224.

as a consequence of thyroid hormone activity. However, no direct proof of this hypothesis has as yet been reported.

Although significant increases in lyso- soma1 enzyme activities have been reported to occur during metamorphosis, little work has been done to demonstrate a n y concomitant qualitative changes in these enzymes. There are, however, two studies which have reported such changes in the nature of lysosomal enzymes. Cathepsin has been found by Weber ('63) to exhibit a lower apparent Michaelis-Menten constant (Km) during late metamorphosis than it does during development. p-glucuronidase has also been studied with respect to several possible qualitative changes OC- curring during tail regression. Price and Frieden ('63) found that there were slight differences in the apparent Km and pH de- pendency of p-glucuronidase preparations taken from developing tadpoles in compar- ison with those from metamorphosing tadpoles. Although these authors suggest that the differences might be due to the presence of modifiers in the preparations, the possibility still remains that there are in fact structural or conformational altera-

1 This investigation was supported by PHS Training grant HD-000032 from the National Institute of Child Health and Human Development.

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216 HELEN ROBINSON

tions in the enzyme during tail atrophy. The demonstration of both qualitative and quantitative changes in lysosomal enzymes during tail regression supports the hypothesis that there is de novo synthesis of specific catabolic enzymes.

In this paper the activity of acid phosphatase in the tail of Xenopus luevis has been examined both during growth and metamorphosis. In addition to the study of activity in the entire tail, the localization of acid phosphatase in ventral fin, dorsal fin, and muscle has been determined and correlations drawn with regional differences in the rate of resorption of parts of the tail. Finally, changes in substrate preference, pH optimum, and apparent Km have been investigated.

MATERIALS A N D METHODS

Tadpoles of Xenopus laevis were raised according to the method of Nieuwkoop and Faber ('56). They were maintained in large plastic bins in a constant temperature room at 22"C, and the well water in which they were kept was changed frequently. At each changing they were fed a freshly prepared suspension of nettle powder in water.

Tadpoles were selected for experiments on the basis of tail length, by extending the tail over a piece of ruled graph paper and measuring by eye to the nearest milli- meter from the tail-body juncture to the tip, Measurements on a large number of tadpoles just prior to the onset of metamorphosis (signaled by eruption of the forelimbs), yielded an average maximum tail length of 36.0 mm with a maximum deviation of 2.0 mm. In all experiments the degree of tail growth or atrophy was expressed as a percent of the average maximum tail length. For Xenopus, this method of establishing the extent of growth or metamorphosis has proved to be more sat- isfactory than the use of a hindlimb/tail ratio or the developmental stages of Nieuw- koop and Faber ('56).

Tissue homogenates were prepared from tails amputated immediately posterior to the anal canal. For studies on enzyme activity in different regions of the tail, the tail was dissected into the dorsal fin, ventral fin, and muscle, and each part was

homogenized separately. A hypotonic buffer system was used during homogeniza- tion to insure release of bound enzyme. No further release of enzyme was effected by sonication or repeated freeze-thawing. Pro- tein concentration was determined by the method of Lowry et al. ('51), and enzyme activity was calculated both in terms of total activity per tail and specific activity per unit of protein. Acid phosphatase activity was assayed by two methods using different substrates.

The assay procedure using p-nitrophenyl phosphate (nPhP) as substrate is a modification of the technique of Bessey et al. ('46). Tail tissues were homogenized for one minute at 1000 rpm in 0.2 ml 0.01 M phosphate buffer pH 7.4 containing 0.001 M EDTA. This homogenate was appropri- ately diluted and then 5 0 4 was added to a reaction mixture containing 0.125 ml 5.0 mM nPhP and 0.125 ml 0.01 M citrate buffer pH 4.8. After incubation for 30 minutes at 37"C, the reaction was stopped by addition of 1.25 ml 0.1 N NaOH, and the absorbance was then measured spectro- photometrically at 410 mp and compared with a p-nitrophenol standard. The assay method was checked to insure that pH was optimal, that the rate was proportional to the amount of enzyme added, and that the substrate concentration was not limiting. Controls for each assay included both a reagent blank and a sample blank. En- zyme activity was expressed as pmoles p- nitrophenol released/hour either per tail or per unit of protein.

The second assay for acid phosphatase with p-glycerophosphate (bGP) as substrate was a modification of the method of de Duve et al. ('55). The tail was homogenized in 0.01 M sucrose containing 0.001 M EDTA. After appropriate dilution, 0.1 mi homogenate was added to a reaction mixture of 0.4 ml 0.125 M bGP and 0.5 ml 0.1 M acetate buffer pH 4.8. This mixture was incubated for 90 minutes at 37"C, and the reaction was stopped by addition of 1.0 ml 18% trichloroacetic acid (TCA). After low speed centrifugation, an aliquot of the supernatant was removed for phosphate determination according to the method of Fiske and Subbarow ('25). The

ACID PHOSPHATASE DURING

amount of inorganic phosphate that could be detected by this colorimetric assay ranged from 0-14 pg. As with the other assay method, all conditions were optimal, and reaction kinetics were zero order. Re- agent and sample blanks were prepared as controls, and enzyme activity was expressed as pmoles phosphate releasedl hour per tail or per unit of protein.

The apparent Km was determined by varying the concentration of nPhP from 2-10 mM and the concentration of bGP from 1-50 mM. Tails from tadpoles in different stages of development and metamorphosis were used to determine whether the apparent Km remains constant. Apparent Kms were calculated from double recipro- cal plots. In addition, the pH optima of both assay systems were tested.

5.1

4.c

3.c

2.0

1.0

ANURAN METAMORPHOSIS 217

RESULTS

The specific activity (SA) of acid phosphatase of whole tail homogenates from tadpoles during development and metamorphosis is depicted in figures l and 2. Figure 1 shows the SA determined by assay with nPhP as substrate, and figure 2 shows the SA determined by assay with bGP. All points reflect assays done on individual tadpoles, hence the amount of scatter indicates the degree of individual variation. The pattern of activity is the same regardless of which assay is used. SA remains constant during development and the initial stages of metamxphosis, but when the tail is approximately 80% of the average maximum length (Lmax). the SA begins to increase sharply. By the time the tail has regressed to 20% Lmax,

. .

0

0

a. 8 . 8

I . I I I I 1 t 40 20 0 6 0 00 100 80 60 40

O/o MAXIMUM TAIL LENGTH Fig. 1 Specific activity of acid phosphatase of whole tail homogenates during growth

and metamorphosis, assayed with nPhP as substrate. The arrow indicates the onset of metamorphosis.

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HELEN ROBINSON

e * e

I I I 1

40 60 80 too 80 60 40 20 0

% MAXIMUM TAIL LENGTH Fig. 2 Specific activity of whole tail homogenates during growth and metamorphosis,

assayed with bGP as substrate. The arrow indicates the onset of metamorphosis.

the SA has increased sixfold according to the nPhP assay, and tenfold according to the bGP assay. The SA is consistently greater in absolute terms when assayed with nPhP as substrate than with bGP.

The total activity per tail (TA) of acid phosphatase from the same series of homogenates assayed by both methods is shown in figures 3 and 4. Again the same pattern of change is found regardless of substrate used, In both cases there is a linear increase in TA during development which continues through the onset of metamorphosis. When the tail is about 609% Lmax the TA levels off, and when the tail has atrophied to about 40% Lmax it begins to decline slowly. Although there is considerable scatter, i t is clear that the TA rises after the onset of metamorphosis and then maintains a high level through most of tail resorption.

Values for specific and total activities of acid phosphatase in different parts of the tail during development and early meta-

morphosis are given in table l . During the first five days of metamorphosis the overall tail length decreases by less than 15% of the Lmax. The dorsal fin, which is the first part of the tail to be resorbed, under- goes considerable shrinkage by the fifth day after onset of metamorphosis (E + 5 ) . The SA of acid phosphatase in the dorsal fin at E + 5 is significantly greater, P < 0.05,2 than it is at onset (E + 0) , and nearly double that in the dorsal fin during development (D) . During this period of early metamorphosis the SA of the ventral fin does not change significantly, nor does the ventral fin undergo any ap- preciable atrophy. The SA of the muscle by the third day after onset ( E + 3) rises significantly, P < 0.01, over the value found during development. At all stages tested, the SA is found to be highest in the ventral fin, followed by the dorsal fin and then the muscle. TA is always found to be high-

2 Student's t test.

ACID PHOSPHATASE DURING ANURAN METAMORPHOSIS 219

a 3.0

=I a

X \

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0’ 2.0 z w I Q 0 [L

k 1.0

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f

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40 60 80 100 a0 60 40 20 0

% MAXIMUM TAIL LENGTH Fig. 3 Total activity of whole tail homogenates during growth and metamorphosis,

assayed with nPhP as substrate. The arrow indicates the onset of metamorphosis.

est in the muscle, then the ventral fin and then the dorsal fin. After day E + 5, the tail shortens dramatically and resorption of the dorsal fin is rapidly completed. Atro- phy of the ventral fin accompanies regression of the muscle and overall tail short- ening.

The apparent Kms for both substrates are shown in figures 5 and 6. Whole tail homogenates from several stages during late development and early metamorphosis were tested in order to detect any overall pattern of change. The apparent Kms €or both substrates remain constant through development and early metamorphosis. During this time, however, the apparent Km for bGP is approximately five times greater than that for nPhP. At the point during tail regression when the length is between 60 and 70% Lmax, the apparent Km for nPhP increases nearly twofold, and that for bGP falls to slightly less than half its original value. In both cases these new levels are maintained throughout the rest of metamorphosis, and it is noteworthy that the apparent Km for nPhP during ad-

vanced tail atrophy is larger than the apparent Km for bGP at this time.

The pH optima of the two assay systems were found to be 4.8 for the nPhP assay, and 5.0-5.8 for the bGP assay. The effect of pH on reaction velocity of the nPhP assay was determined for tadpoles in development, and in early and late metamorphosis. There appears to be no difference between enzyme preparations from tadpoles in early metamorphosis (prior to 80% Lmax) and tadpoles in developing stages, hence these results are grouped together in figure 7. The shape of the pH curve from tadpoles undergoing advanced tail atrophy (10-30% Lmax) is quite different from the shape of the curve obtained from developing and early metamorphosis tadpoles. The entire curve is shifted over into a more acid range during late metamorphosis, and the optimum becomes 4.2 instead of 4.8.

DISCUSSION

The results of this investigation of acid phosphatase activity in the tail of Xenopus tadpoles, indicate that both quantitative

220 HELEN ROBINSON

1.4

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. 0 .

0 . 0 . .

a

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Fig. 4 Total activity of whole tail homogenates during growth and metamorphosis, assayed with bGP as substrate. The arrow indicates the onset of metamorphosis.

TABLE 1

Specific and total activities of acid phosphatase in various regions of the tail during early metamorphosis. Each entry is the mean 5 standard error; number of

determinations in parentheses

Average SA SA SA TA TA TA Stage % Lmax Dorsal fin Ventral fin Muscle Dorsal fin Ventral fin Muscle ~ _ _ _ _

- - - 0.71?0.03 1.90t0.14 0.5050.03 -

(6) (6) (6)

( 6 ) (5) ( 6 ) ( 6 ) ( 5 ) (6)

(4) ( 4 ) (5) (4) (5) (5)

(5 ) (5) (6 ) (6 ) ( 5 ) (5)

(5) (6) (6 ) ( 5 ) (6) (6)

D

E+O 97.1 1.0120.09 1.7450.10 0.5050.04 0.04%0.01 0.19&0.01 1.6750.13

E + l 91.7 1.01?0.08 1.8750.20 0.5050.01 0.04C0.01 0.18-CO.01 1.5920.10

E + 3 86.1 l . l O ? 0.03 1.762 0.13 0.70C 0.01 0.03 C 0.001 0.18k 0.01 1.59k 0.07

E + 5 91.7 1.3020.07 1.99-0.17 0.77t0.04 0.0550.01 0.2150.01 1.87k0.09

and qualitative changes occur in the en- Quantitative measurements of acid phos- zyme during tail regression. Furthermore, phatase activity during development and it is shown that both kinds of changes ap- metamorphosis reveal the same general pear at roughly the same stage of meta- pattern regardless of which assay method morphosis. is used. In absolute terms, however, en-

ACID PHOSPHATASE

10 c

a -

6 '

Km (mM)

2 -

DURING ANURAN METAMORPHOSIS

L

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16

12

Km

r

-

-

. 0 . 0.

0 .* .

J I I

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'/o MAXIMUM TAIL LENGTH

Fig. 5 The apparent Km of acid phosphatase of whole tail homogenates for substrate nPhP during both growth and metamorphosis. The arrow indicates the onset of metamorphosis.

. .

L I , I I 8

6 0 80 100 80 60 40 20 0

O/o MAXIMUM TAIL LENGTH

Fig. 6 The apparent Km of acid phosphatase of whole tail homogenates for substrate bGP during both growth and metamorphosis. The arrow indicates the onset of metamorphosis.

zyme activity determined by the nPhP assay is consistently higher than it is when the bGP assay is used. The simplicity of the nPhP assay and the great reactivity of

acid phosphatase with this substrate rec- ommend this method for studies on small amounts of tissue and tissues with low enzyme content. Results from the bGP assay

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0 0

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20

A

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-

-

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HELEN ROBINSON

B

Fig. 7 Typical Lineweaver-Burk plots for acid phosphatase using the nPhP assay system. Units of S are molar concentration x lo-'. A, developing tail; B, tail in late metamorphosis.

0'

I I 1 I I I I 4 3.5 4.0 4.5 5.0 5.5 6.0 6.5

PH Fig. 8 The effect of pH on reaction velocity using the nPhP assay system with citrate

buffers used for all assays. A different homogenate was used for each determination at a given pH. Open dots represent late metamorphosis tails (L is 10% to 30% of Lmax), closed dots represent developing and early metamorphosis tails (L is 80% to 100% of Lmax).

confirm earlier reports (Weber, '61 ; Eeck- hout, '65) that the enzyme increases significantly during tail resorption.

Increases in enzyme activity in whole tails and in different parts of the tail are found to correspond in time to periods of active tissue regression. The sharp rise in SA of whole tail homogenates occurs during the period of most rapid tail shorten- ing. The increase in SA of the dorsal fin during the early stages of metamorphosis

prior to extensive tail atrophy, corresponds temporally with the specific resorption of this region of the tail at this time. In both these instances, increases in SA are ac- companied by increases in TA per tail or per dorsal fin, indicating that these rises are not merely due to loss of protein.

Qualitative changes in tail acid phosphatase during metamorphosis are clearly demonstrated by the results of the studies on apparent Km and pH optimum. Both of

ACID PHOSPHATASE DURING ANURAN METAMORPHOSIS 223

these enzyme properties remain constant throughout development and very early metamorphosis, as does the SA of the whole tail. Significant changes in these properties occur when the tail is about 60-70% Lmax. There is a strong correlation bc- tween the time of increase in activity in the tail and the shift in apparent Kms for both substrates, although rises in activity are detected slightly before any qualitative change is noted.

Several interesting conditions are reflect- ed by the qualitative changes that take place in acid phosphatase in the tail during metamorphosis. The change in pH optimum toward a more acid pH, and the shift of the pH optimum curve into a more acid range correlate well with a report (Aleschin, '26) that the pH of regressing tails is lower than that of developing tails. The changes in substrate preference of acid phosphatase which occur during tail regression, as represented by changes in the apparent Kms for nPhP and bGP, are comparable to changes in substrate preference of alkaline phosphatase in mouse duodenum reported by Moog et al. ('66) and Moog ('66). In both situations a change in substrate preference of an enzyme is correlated with a developmental change, although the work of Moog has gone further to show that there is an actual conversion from one form of the enzyme to the other which is stimulated rather than blocked by inhibitors of protein synthesis. It might well be the case that acid phosphatase of tadpole tail tissues consists of several forms of the enzyme which are present in different relative amounts during development and metamorphosis, and that the shift in substrate preference re- flects precisely such a conversion in relative abundance from one form to another. Other alternatives can be suggested, such as synthesis of a new form of the enzyme or a conformational change in the enzyme without new synthesis.

Current investigation of the electro- phoretic separation of acid phosphatase from individual tadpole tails on acrylamide gels has demonstrated the presence of two distinct forms of the enzyme during both development and metamorphosis. Densito- metric tracings of gels show that the amount of one form relative to the other

remains essentially constant during late development and early metamorphosis (tail length > 80% Lmax). However, in the late regressing tail this ratio is significantly changed, due primarily to a dramatic increase in the amount of one form with some concurrent decrease in the other. Experiments to determine the relative con- tributions of synthesis and/or conversion in bringing about these changes are in progress.

ACKNOWLEDGMENT

I wish to thank Dr. E. J. Boell for his ad- \-ice and help with this research.

LITERATURE CITED Aleschin, B. W. 1926 Die Aktuelle Reaktion

des Gewebssaftes bei Normaler und Besch- leunigter Metamorphose von Rana temporaria. Biochem. Z., 171: 79-82.

Bessey, A., 0. H. Lowry and M. J. Brock 1946 A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem., 164: 321-329.

Coleman, J. R. 1962 Deoxyribonuclease activities in the development of the leopard frog, Ranu pipiens. Dev. Biol., 5: 232-251.

1963 Acid deoxyribonuclease activity in amphibian metamorphosis. Biochim. Biophys. Acta, 68: 141-143.

de Duve, C., B. C. Pressman, R. Gianetto, R. Wat- tiaux and F. Applemans 1955 Tissue frac- tionation studies. 6. Intracellular distribution patterns of enzyme in rat-liver tissues. Bio- chem. J., 60: 604-617.

Eeckhout, Y. 1965 Contribution a l'etude de la metamorphosi caudale des amphibiens anoures. These Univ. cathol. Louvain. Fac. Sci.

Fiske, C. H., and Y. Subbarow 1925 The colorimetric determination of phosphorus. J. Biol. Chem., 66: 375-400.

Kubler, H., and E. Frieden 1964 The increase in p-glucuronidase of the tadpole tail during anuran inetamorphosis and its relation to lysosomes. Biochim. Biophys. Acta, 93: 635-643.

Lapiere, C. M., and J. Gross 1963 Mechanisms of hard tissue destruction. Publ. No. 75. Am. Assoc. Advance. Sci., Washington, D. C., pp. 663-694.

Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem.,

Moog, F. 1966 The regulation of alkaline phos- phatare activity in the duodenum of the mouse from birth to maturity. J. Exp. Zool., 161: 353-

1996 The multiple forms of alkaline phosphatase in the small intestine of the young mouse. Biochim. Biophys. Acta, 113: 336-349.

Nieuwkoop, P. D., and J. Faber 1956 Normal Table of Xenopus laevis (Daudin). North Hol- land Publishing Co., Amsterdam.

193: 265-275.

368. Moog, F., H. R. Vire and R. D. Grey

224 HELEN ROBINSON

Price, S., and E. Frieden 1963 p-glucuronidase from amphibian tails. Comp. Biochem. Physiol., 10: 245-251.

Tata, J. R. 1966 Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture. Dev. Biol., 13: 77-94.

Weber, R. 1957a On the biological function of cathepsin in tail tissue of Xenopus larvae. Ex- perientia, 13: 153-155. - 1957b Die Kathepsinaktivitat im Schwanz von Xenopuslarven wahrend Wachstum und Metamorphose. Rev. Suisse Zool., 64: 326-335.

1963 Ciba Foundation Symposium on Lysosomes. Little, Brown and Company, Boston, Massachusetts, pp. 283-200.

1964 Ultrastructural changes in regressing tail muscles of Xenopus larvae at meta morphosis. J. Cell Biol., 22: 481-487.

1965 Inhibitory effect of actinomycin D on tail atrophy in Xenopus larvae at metamorphosis. Experientia, 21 : 665-666.

Weber, R., and B. Niehus 1961 Zur Activitat der sauren Phosphatase im Schwanz der Xen- opuslarven wahrend Wachstum und Metamor- phose. Helv. Physiol. et Pharmacol. Acta, 19: 103- 1 17.

ERRATUM

The values on the ordinate on figure 5, page 221, and the values on the abscissas on figure 7, page 222, should be multi- plied by 0.4.

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