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The inncrease in β-glucuronidase of the tadpole tail during anuran metamorphosis and its relation to lsysosomes
BIOCI-IIMICA ET BIOPHYSICA ACTA
635
BBA 2 5 1 3 4
T H E I N C R E A S E IN f l - G L U C U R O N I D A S E O F THE TADPOLE TAIL DURING ANURAN METAMORPHOSIS A N D ITS R E L A T I O N T O L Y S O S O M E S H E L G A K U B L E R * AND E A R L F R I E D E N
Department of Chemistry and The Institute of Molecular Biophysics, Florida State University, Tallahassee, Fl. (U.S.A .) (Received April 13th, 1964)
SUMMARY
The activity of soluble and particle-bound fl-glucuronidase (fi-D-glucuronide glucuronohydrolase, EC 3.2.1.31 increased over 3o-fold in the regressing tadpole tail during spontaneous metamorphosis. The total activity of fl-glucuronidase per tail also appears to go up 3 times during tail resorption. The increase in soluble enzyme activity occurs simultaneously in the fin, skin and muscle at the beginning of tail resorption. During induced metamorphosis, the soluble fl-glycuronidase activity increases to only onethird of that found during spontaneous metamorphosis and the protein concentration doubles. From the present data it has not been possible to use the distribution of and the changes in fl-glucuronidase activity as evidence for the existence of lysosomes in the tadpole tail.
INTRODUCTION
Anuran metamorphosis represents the developmental process of the transformation of an aquatic larva to a terrestrial frog. Classical studies of the morphological changes during the process of metamorphosis have culminated in the work of TAYLOR AND KOLLROS 1, and much of the current biochemical information has been summarized by BENNETT AND FRIEDEN2. The regressing tadpole tail is an excellent model system for studying characteristic biochemical changes during the histolysis of a tissue. The possibility that tail resorption is an expression of the controlled activity of intracellular catabolic enzymes has long been suspected 2. Some of the proposed mechanisms concerning the regulation of the activity of the intracellular enzymes have been summarized recently 3. In 1955, DEDtlVE~ proposed that numerous lytic enzymes might be contained in an intracellular entity, the lysosome. An intriguing idea concerning the mechanism of cellular dissolution, resulting in tail disappearance, is that it might be caused by the controlled disintegration of the tail lysosomes. This would cause a release of the Abbreviation: Ta, 3',3,5-triiodothyronine. * Present address: Department of Physiological Chemistry, Medical Science Building, The University of Wisconsin, Madison, Wisc.
Biochim. Biophys. Acta, 93 (1964) 635 643
636
H. KUBLER, E. FRI ED EN
hydrolytic enzymes and the eventual self digestion of the affected cellsa, 5. There is little doubt that as the tail recedes all structural elements disappear and the enzymes claimed to be lysosomal are retained (Table IV). In order to study these tissue changes further, we have examined the activity of the lysosomal enzyme, ~-glucuronidase (/3-D-glucuronide glucuronohydrolase, EC 3.2.I.3I), during spontaneous and induced metamorphosis. A previous report by PRICE AND FRIEDE• 6 established that/3-glucuronidase did not appear to undergo any detectable molecular alteration during metamorphosis. MATERIALS AND METHODS
Animals This study was performed exclusively on Rana grvlio tadpoles collected at different times of the year and from ponds in the Tallahassee, Florida area. They were fed a diet of collard greens and Bacto-Veal and maintained in an aireonditioned laboratory. The animals used in each series of experiments were all of similar size and leg development and from the same source, although a common genetic origin cannot be guaranteed. Induced metamorphosis As described earlier 7, the tadpoles were injected with IO-~/,moles (6. 7/,g of the sodium salt) T 3 per g of tadpole. Controls were injected with amphibian Ringers solution (pH 8.7). The T a was dissolved in Ringers with sufficient sodium hydroxide to dissolve it and all solutions used were freshly prepared. Protein determination The concentration of soluble protein in the Io5ooo x g supernatant was determined by the method of LOWRY et al. 8 using crystalline serum albumin as a standard. [3-Glucuronidase assay The enzyme assay is a modification of the procedure of FISHMANet al. 9 To o.Io ml of a properly diluted sample, 0.80 ml of o.Io M acetate buffer (pH 4.5) and o.Io ml of a O.OLOM phenolphthalein ]3-glucuronide solution were added. After the samples were incubated at 37 ° for 60 rain, 5.0 ml of 0.20 M glycine buffer (pH lO.4) in 0.2 M NaC1 was added. For samples with low activity 2.0 ml of glycine buffer (pH 11.2) was added. This stopped the reaction and produced the red color of the phenolphthalein liberated by the enzyme. Absorbancy was measured at 552 in/, in a Beckman DU spectrophotometer. The enzyme activity is expressed in units per g wet weight of tail tissue. A unit (equivalent to one FISHMAN9 unit) is defined as being equivalent to I / , g of phenolphthalein liberated in I h at pH 4-5 and 37 ° from o.ooi M phenolphthalein fi-glucuronide solution in a total volume of I ml. Specific activity is defined as the ratio of the units of enzyme activity to the protein content of the enzyme solution in mg per g of wet tissue weight. At the wavelength at which the readings were taken, no fading occurred within 15 min. Blanks contained all components of the reaction except the substrate. Under the above conditions the liberation of phenolphthalein is a linear function of time and of enzyme concentration. However, it should be noted that 0.25 M sucrose produces 25-30 % inhibition of enzyme acBiochim. Biophys. ,4cla, 93 (I964) 635-643
LYSOSOMES AND TADPOLE TAIL fl-GLUCURONIDASE
637
tivity 5. Since all assays were performed in o.25 M sucrose, the inhibition is assumed to be constant in all cases and no correction was made.
Preparation of the variousfractions for fl-glueuronidase measurements For the determination of soluble fl-glucuronidase activity of entire tails, six tails of tadpoles or froglets were homogenized in four times their weight of 0.25 M sucrose containing 0.o2 M Tris buffer (pH 7.2). The time of homogenization was 30 sec in a Virtis blendor and I rain in a P o t t e r - E l v e h j e m type homogenizer, equipped with a conical pestle. After differential centrifugation for IO min at 60o × g and 9600 × g and 30 rain at lO5 ooo x g (Spinco Model L Rotor No. 4o), the lO5 ooo × g supernatant was analyzed for the activity of soluble fl-glucuronidase. The activity of soluble fl-glucuronidase was determined on the different parts of the tail from the same animal: the skin, the fin (from which the skin was removed) and the muscular portion. About 7 ° mg of each tissue was homogenized in a small P o t t e r - E l v e h j e m type homogenizer for 30 sec. After differential centrifugation using the swinging bucket rotor, SW 39, specific activity of fl-glucuronidase was determined as described above. For the determination of fl-glucuronidase (soluble plus bound) Triton X - I o o (Robin and Haas Company, Philadelphia, Pa.) was added to the 6o0 × g and 9600 × g supernatants in order to disrupt any intact particles. The residues containing the lysosomal fraction (960o × g residue) and the microsomal fraction (lO5OOO x g residue) were suspended in 0.5 ml 0.25 M sucrose and o.40 ml o.Io N acetate buffer (pH 4-5) in 0.25 M sucrose and o.Io ml 1 % Triton in 0.25 M sucrose. The mixture was incubated in 37 ° for 8 rain, cooled for 2 rain and centrifuged for 30 min at 1o5000 × g in order to release the enzyme. The enzyme activity in the supernatant was measured. The resulting pellet was also tested for enzyme activity to insure a complete release of the enzyme. RESULTS
Soluble fl-glucuronidase activity during spontaneous metamorphosis The increase in the specific activity of soluble fl-glucuronidase during spontaneous metamorphosis is illustrated in Fig. I. Each point represents 3 groups and each group contains 6 tails. The developmental stage of the tadpoles or froglets is usually expressed as the ratio of hindleg length to tail length. Fig. I shows that there is no increase in specific activity at the stage when tadpoles have no legs including the time when the hindlegs and frontlegs are breaking through and there is still considerable growth. At a hindleg to tail ratio in the range o.9-1.1 (initial tail length 65 mm), tail reduction starts and an increase in the specific activity of fl-glucuronidase is observed. At an advanced stage of metamorphosis, hindleg to tail ratio of 12 (final tail length 5 ram), a 34-fold increase in the specific activity of tail fl-glucuronidase is achieved. Since at this stage the tail is I / I I of its original weight, these data suggest that there is a 3-fold increase in the total amount of soluble fl-glucuronidase in the tail. Likewise, the large increase in specific activity cannot be attributed to a decrease in soluble protein. The amount of soluble protein decreases slightly at first and then remains constant (Fig. 4). As tail resorption starts, the fins are absorbed first. Therefore, preparations of Biochim. Biophys. Acta, 93 (1964) 635-643
H. KUBLER, E. FRIEDEN
638
skin, skinless fin and muscle were studied for changes in fl-glucuronidase specific activity in these various parts of the tail. The results (Fig. 2) show that while the initial activities are different, the increase in specific activity of soluble fl-glucuronidase in these three tissues occurs simultaneously with the onset of the tail resorption. SPECIFIC
ACTIVITY
g
OF
"o
"~ 15C
SKIN
zo
IO0i
ili
ID
,7
• *4
I
0
2
,
I
4
,
1
,
I
6 8 LEG/TAIL
l
1 I0
,
I~j__L
I
0.5
12
Fig. I. The increase of specific activity of soluble fl-glucuronidase during s p o n t a n e o u s m e t a m o r phosis. The e n z y m e activity was determined in the s u p e r n a t a n t after centrifugation at 1o5oo0 × g. The horizontal axis refers to the ratio hindleg length: tail length; the vertical axis refers to the specific activity of the enzyme in t e r m s of lO u n i t s of enzyme a c t i v i t y per m g protein per i g of tail (wet).
0.6
....... L___
0.7 0.8 LEG/TAIL
t
1
0.9
1.0
Fig. 2. Tile specific activity of soluble fl-glucuronidase in skinless fin, skin and muscle at the beginning of s p o n t a n e o u s m e t a m o r p h o s i s . Each point represents six individual tails. Coordinates as in Fig. i.
TABLE I ACTIVITY OF
SOLUBLE
fl-GLUCURONIDASE
IN
UNITS
PER
g
OF
TADPOLE
TAIL
A, induced metanmrphosis Hindleg length when injected and temperatures at ~kick the tadpoh's arc kept
0. 5 cm (kept at 26 °) 4.0 cm (kept at 23 _7 2~)
4.5 days:
5.5 days,"
7 days;
40 % tail reduction
48 % tail reduction
72 % tail reduction
i6o-i9o
3oo-33 o --
dead 132o-138o
B, spontaneous metamorphosis H indlcg length and temperatures at which the tadpoles are kept
0. 5 cm (kept at 26 "~) 4.0 cm (kept at 2 3 :: 2 )
4.5 oo tail reduction
930-97 ° -
72 % tail reduction
-18oo-19oo
fi-Glucuronidase activity during induced metamorphosis When metamorphosis is stimulated by Ta, the increase in soluble fl-glucuronidase activity is about i/a of the increase observed for the same amount of tail reduction 13tochim. t3iophys. Acta, 93 (I964) 035 043
639
LYSOSOMES AND TADPOLE TAIL ~-GLUCURONIDASE
occurring during spontaneous differentiation. As shown in Table I, when a later stage is used for induction, the enzyme activity approaches that of the corresponding level of tail reduction in normal metamorphosis. _.J <
I-
z ~ 5o 0 w I1.
/
/
I'--I. . . . .
/v--~---- t
-I
//
m 30 -
m
I I I CONTROL
?--I--~"
_.1 o u~
T3
z
~ 30 rn 0
~,o
i0
I
0
,
I
2
p
I
,
4
DAYS AFTER
I
6 T5
Fig. 3- The increase of soluble protein (supern a t a n t after 10500o x g centrifugation) per g of tail (wet) in induced m e t a m o r p h o s i s .
E
SPONTANEOUS [
L
~0% DECREASE
I
t
,
4o% IN
TAIL
,
I
70% LENGTH
Fig. 4. A c o m p a r i s o n of the a m o u n t of soluble p r o t e i n in the 105000 × g s u p e r n a t a n t in spont a n e o u s a n d induced m e t a m o r p h o s i s as a function of the percent decrease in tail length. A 70% decrease corresponds to a leg: tail ratio of a b o u t 2. 5 .
It was suspected that the large increase in cathepsin (EC 3.4.4.9) activity 1°'2° might result in a solubilization of tail protein. Therefore, we examined the change in the concentration of soluble tail protein during induced metamorphosis. Fig. 3 shows that after T 3 injection a 60 % increase in soluble protein occurs. As shown in Fig. 4 the amount of soluble protein is twice that in spontaneous metamorphosis at the same decrease in tail length. This increase in soluble protein is not dependent on the stage of the tadpole used for injection. Thus, the change in specific activity of tail fi-glucuronidase would be reduced b y a factor of two if expressed on the basis of soluble protein content.
Experiments testing the possible lysosomal nature of tail fl-glucuronidase Since lysosomes are believed to be highly sensitive to rupture by physical forces, tadpole and froglet tails were homogenized in both isotonic and hypotonic medium for varying periods. From the data in Fig. 5 it appears that no additional enzyme is solubilized or released from tadpole tails after 15 sec of treatment in a "Virtis" blendor. If the tail contained an appreciable amount of enzyme not released by homogenization in 15 sec, the appearance of this enzyme in tail supernatants would have been expected. In the froglet, fl-glucuronidase is continuously released for 45 sec. If tail fl-glucuronidase is lysosomal and the lysosomes break down as metamorphosis proceeds, the release of lysosomal bound fi-glucuronidase should increase the amount of soluble fl-glucuronidase during tail resorption, while the total amount of enzyme activity remains constant. To measure the amount of bound activity the enzyme was released from the mitochondrial-lysosomal fraction, the residue at 9600 :.7 g, b y incubation with o.I % Trition X-Ioo. The results summarized in Table II show about a thirty-fold increase of fl-glucuronidase activity in the "particle" bound tYiochim. Biophvs. Acta, 93 (1964) 635-643
640
H. KUBLER,
E.
FRIEDEN
and soluble enzyme per g of tail tissue. The total increase per tail in both fractions is three-fold. Thus, the data indicate that there is no direct transformation of fl-glueuronidase from the particle bound to the soluble state in the autolyzing tail. Q hi
N _J m
=, 0 (/3
FROGLET
30C
_
Z
200
g IOC :3
20
40
SECONDS
60
IN BLENDER
Fig. 5. Tadpole and froglet tails were blended in o. 25 M sucrose or distilled water for varying lengths of time in the "Virtis" blendor. After centrifugation at 1o50oo × g for 3o rain the resulting supernatant was assayed for fl-glucuronidase. TABLE I1 THE
SOLUBLE fl-GLUCURONIDASEACTIVITY OF TADPOLE TAILS DURING SPONTANEOUS METAMORPHOSIS
INCREASE
IN BOUND
AND
Each number represents the average of 4 experiments.
Stage of tadpole
Ratio of lengths hindleg/taiI
Tail weight ( g)
Releasable fl glucuronidase activity from "Lysosomal" fraction (units) One tail
Early Intermediate Late
0.2 o.9 7.0
0.82 o.73 0.073
29 65 84
I.o g of tail
35 89 i ~5o
Soluble fl glucuronidase activitYsupernatantinio 5 ooo × g (units) One tail
r.o g of tail
85 246 226
lO3 337 3092
Intracellular localization of fl-glucuronidase in tadpole tails The intracellular localization of tadpole fl-glucuronidase was studied in more detail. Tail homogenates were subjected to differential centrifugation to yield a nuclear fraction (600 x g residue), a mitochondrial-lysosomal fraction (9600 ; g residue) a n d a m i c r o s o m a l f r a c t i o n (lO 5 ooo ~, g residue). T h e t o t a l a c t i v i t y in t h e r e s p e c t i v e s u p e r n a t a n t s a n d t w o of t h e r e s i d u e s was d e t e r m i n e d a f t e r t r e a t m e n t w i t h o . I o % T r i t o n X - I o o to release b o u n d e n z y m e f r o m all particles. As s h o w n in T a b l e III, t h e a c t i v i t y r e m a i n e d p r e d o m i n a n t l y s o l u b i l i z e d in e a r l y t a d p o l e s w h i c h were at t h e s t a g e p r i o r t o t h e m a j o r i n c r e a s e in t h e specific a c t i v i t y of fi-glucuronidase. A b o u t 20 % of t h e a c t i v i t y s e d i m e n t e d w i t h t h e m i t o c h o n d r i a l - l y s o s o m a l f r a c t i o n a n d I 0 % in t h e m i c r o s o m a l f r a c t i o n . I t was possible to a c c o u n t for t h e d e c r e a s e in t h e a c t i v i t y of e a c h s u p e r n a t a n t r e p o r t e d in T a b l e I I I b y d e t e r m i n i n g t h e r e l e a s a b l e e n z y m e w h i c h a c c o m p a n i e d e a c h residue. F o r e x a m p l e , at an l e g : t a i l r a t i o of 0. 9 t h e a c t i v i t y of t h e s u p e r n a t a n t f r o m Biochim. Biophys. Acta, 93 (I964) 635-643
641
LYSOSOMES A N D T A D P O L E T A I L ~ - G L U C U R O N I D A S E TABLE
III
MAXIMUM RELEASABLE ~-GLUCURONIDASE ACTIVITY OF VARIOUS TAIL FRACTIONS
E a c h number represents IO experiments. Hindleg:tail = 0.2
Hindleg:tail = 0. 9
Hindleg:tail -
x.2-
L3
Supematant after centrifugation at
Units/g
S .D.
Units/g
S.D.
Units/g
S.D.
600 × g 96oo × g lO 5 ooo x g
119 94 81
0. 3 o.3 0. 3
312 253 217
5.4 5.3 5.2
591 485 432
4 .1 4 .o 4.0
the 96oo × g centrifugation had decreased by 59 units and precisely 59 units of enzyme activity were obtained from the Triton treated residue from this fraction. The next fractionation resulted in a decrease of 36 units and 35 units were recovered from the microsomal residue. Satisfactory recoveries of the enzyme in the appropriate residue were obtained in every experiment reported in Table III. DISCUSSION
The large increase in /~-glucuronidase activity establishes this enzyme as one of a group of tail enzymes whose specific activity increases significantly during anuran metamorphosis. As summarized in Table IV, numerous hydrolytic enzymes increase many-fold during the transition of tadpole to frog. Next to the thirty-four-fold increase in /~-glucuronidase reported here, the most impressive activity increase is shown by the intracellular protein hydrolase, cathepsinl°, ~°. A high level of acid phosphatase (EC 3.1.3.2) activity in the regressing Rana pipiens tadpole tail has been demonstrated cytochemically by NOVIKOFFll. We regard it as more than coincidence that all of these enzymes, except collagenase (EC 3.4.4.I9), have been described as lysosomal by DEDUVE 12 and NOVIKOFF13. However, it must be emphasized that several enzymes usually considered lysosomal, e.g. RNAase (EC 2.7.7.16) and lipase TABLE
IV
CHANGES IN ACTIVITIES OF TADPOLE-TAIL ENZYMES DURING METAMORPHOSIS
Several tail e n z y m e s are reported ~ to have no significant increase in a c t i v i t y i n c l u d i n g R N A a s e , lipase, and a protein hydrolase (at p H 8.7). A T P a s e decreases in a c t i v i t y 19 a b o u t 5 I o - f o l d . Relative increase in activity*
Enzyme
fl-Glucuronidase CathepsinX0,20 Collagenase zl Acid phosphatasen,~2, za A protein hydrolase (optimum p H 4.2) 24 Dipeptidases (several kinds)24, 25 D N A a s e ( E C 3 . I . 4 . 5 ) 26
Alkaline phosphatase ( E C 3 . I . 3 . I . ) 2 a
34 22 io 4 IO 9 3-7 5 3
* Relative increase in a c t i v i t y is defined as the ratio of the e n z y m e a c t i v i t y at tile conclusion of spontaneous or induced metamorphosis to the a c t i v i t y found in the early tadpole. Biochim.
B i o p h s , s. A c / a ,
93 (1964) 6 3 5 - 6 4 3
642
H. KUBLER, E. FRIEDEN
(EC 3.I.I.3), do not increase in activity. The possible participation of lysosomal enzymes in the autolysis of the tadpole tails during anuran metamorphosis has been suggested earlier 3,5. However, these data do not yet offer support for the existence of lysosomes in the tadpole tail or the presence of tail fi-glucuronidase in a discrete cell particulate. Assuming that the properties of tadpole tail "lysosomes" are identical with those of rat-liver lysosomes, the "lysosomal" enzymes should be particle-bound and released by detergents or by prolonged homogenization. With tadpole tails, prolonged homogenization did not release particle-bound enzyme, but soluble activity was obtained from froglet tails by this procedure (Fig. 5). It is still possible that the lysosomes are first formed when the tail reduction starts, or that the enzyme is present in an inactive form in tadpole tail "lysosomes" and therefore not detectable, or that the lysosomes are destroyed by the mild homogenization process. No evidence for the appearance of an activator or the loss of an inhibitor was obtained after mixing with the various fractions of supernatants prepared from homogenates of tadpoles and froglet tails. The sum of the activity of the corresponding fractions was always obtained. The amount of activity did not change after subjecting supernatants to dialysis. These results do not exclude the possibility that an activator or an inhibitor might have been destroyed during the preparation of the tail tissue. PRICE AND FRIEDEN6 compared the properties of partially fractionated tadpole and froglet fl-glucuronidase and found no significant differences between these two enzyme preparations. It was observed that there occurs a three-fold increase in total activity per tail in both the particulate "lysosomal" (residue 96oo ~'
LYSOSOMES AND TADPOLE TAIL ~-GLUCURONIDASE
643
enzyme and therefore not the most favorable enzyme to test the lysosome mechanism of tadpole tail autolysis. I t was observed that the change in fl-glucuronidase activity was greater during spontaneous than induced metamorphosis for a corresponding tail reduction. There is ample evidence that T 3 treatment does not always produce an effect identical with those observed during the slower apparently more ordered changes in spontaneous metamorphosis. Strong morphological as well as biochemical differences have been noted earlier 3. Considerable variation has also been observed in enzymic responses. Tail-ATPase activity declines five- to ten-fold during spontaneous metamorphosis, but increases about 1/3 after T3 treatment 17. In m a n y other biochemical responses during metamorphosis such as the increase in serum proteins TM and the changes in hemoglobin m, comparable Ta effects are even more difficult to demonstrate. ACKNOWLEDGEMENT
This research was supported in part by grant C-3oo6 from the U.S. Public Health Service. This is paper No. 19 in a series on the biochemistry of amphibian metamorphosis. REFERENCES 1 C. A. TAYLOR AND J. J. KOLLROS, Anat. Record, 94 (1946) 72 T. P. BENNETT AND E. FRIEDEN, in M. FLORKIN AND H. S. MASON, Comparative Biochemistry, Vol. 4, P a r t B, Academic Press, N e w York, I962, p. 483 . 3 E. FRIEDEN, Principal Biochemical Changes During Amphibian Metamorphosis. Proceedings of the Second International Conference on Congenital Malformations, July, 1963, New York (in the press). 4 C. DEDuvE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS, Biochem. J., 60 (1955) 604. s C. DEDuvE, Funhtionelle und Morphologische Organization der Zelle, \Viss. Konf. der Ges. dtsch. Naturf. u n d Arzte, Springer-Verlag, 1963, p. 209. 6 S. PRICE AND E. FRIEDEN, Comp. Biochem. Physiol., IO (1963) 245. 7 E. FRIEDEN AND G. W. WESTMARK, Science, 133 (1961) 1487. 8 0 . I-1. LOWRY, N. J. ROSEBOROUGH, A. C. FARR AND B. J. RANDALL,J. Biol. Chem., 193 (1951) 265, 9 \¥. H. FISHMAN, B. SPRINGER AND R. BRUNETTI, J. Biol. Chem., 173 (1948) 44910 R. WEBER, Induktion und Morphogenese, 13. Coloq. der Ges. f. Physiol. Chemic, Mosbach, Springer-Verlag, 1962, p. 235. 11 A. B. NOVIKOFF, in D. RUDNICK, Developing Cell Systems and Their Control, Ronald Press, New York, 196o, p. 167. 12 C. DEDUVE, in T. HAYASHI, Subcellular Particles, Ronald Press, New York, 1959, p. 128. 13 A. B. NOVIKOFF, in J. BRACHET AND A. E. MIRSKY, The Cell, Vol, 2, Academic Press, N e w York, 1961, p. 299. 14 K. PAIGEN, Exptl. Cell Res., 25 (1961) 286. 15 N. PASETTO, Inform. Med. Sez. Clin. Sci., 19 (1955) 287. 16 R. WEBER, in A. U. S. DE REUCK AND M. P. CAMERON (Eds.),CibaFound. Syrup. on Lysosomes, J. A. Churchill, L o n d o n , 1963, p. 282. 17 E. FRIEDEN AND H. MATHEVVS, Arch. Biochem. Biophys., 73 (1958) lO7. is A. E. HERNER AND E. FRIEDEN, J. Biol. Chem., 235 (196o) 2845. 19 C. D. TRADER, J. S. \VORTHAM AND E. FRIEDEN, Science, 139 (1963) 918. 2o R. WEBER, Experientia, 13 (I957a) 153. 21 j. GROSS AND C. M. LAPIERE, Proc. Natl. Acad. Sci., 48 (1962) l o l 4. 22 R. WEBER AND B. NIEHUS, Helv. Physiol. Acta, 19 (1961) lo 3. 23 T. YANAGISAWA, Rept. LiberalArts Sci. Fac. Shizuoka Univ. Nat. Sci., 4 (1953) 20; 5 (1954) 3324 E. URBANI, Rend. Ist. Lombardo Sci. Lettere B, 92 (1957) 69. 25 A. M. ZACCHEI, Rieerca Sci. suppl., 24 (1954) 1489. 2~ J. R. COLEMAN, Biochim. Biophys. Acta, 68 (1963) 141.
Bioehim. Biophys. Acta, 93 (1964) 635-643
635
BBA 2 5 1 3 4
T H E I N C R E A S E IN f l - G L U C U R O N I D A S E O F THE TADPOLE TAIL DURING ANURAN METAMORPHOSIS A N D ITS R E L A T I O N T O L Y S O S O M E S H E L G A K U B L E R * AND E A R L F R I E D E N
Department of Chemistry and The Institute of Molecular Biophysics, Florida State University, Tallahassee, Fl. (U.S.A .) (Received April 13th, 1964)
SUMMARY
The activity of soluble and particle-bound fl-glucuronidase (fi-D-glucuronide glucuronohydrolase, EC 3.2.1.31 increased over 3o-fold in the regressing tadpole tail during spontaneous metamorphosis. The total activity of fl-glucuronidase per tail also appears to go up 3 times during tail resorption. The increase in soluble enzyme activity occurs simultaneously in the fin, skin and muscle at the beginning of tail resorption. During induced metamorphosis, the soluble fl-glycuronidase activity increases to only onethird of that found during spontaneous metamorphosis and the protein concentration doubles. From the present data it has not been possible to use the distribution of and the changes in fl-glucuronidase activity as evidence for the existence of lysosomes in the tadpole tail.
INTRODUCTION
Anuran metamorphosis represents the developmental process of the transformation of an aquatic larva to a terrestrial frog. Classical studies of the morphological changes during the process of metamorphosis have culminated in the work of TAYLOR AND KOLLROS 1, and much of the current biochemical information has been summarized by BENNETT AND FRIEDEN2. The regressing tadpole tail is an excellent model system for studying characteristic biochemical changes during the histolysis of a tissue. The possibility that tail resorption is an expression of the controlled activity of intracellular catabolic enzymes has long been suspected 2. Some of the proposed mechanisms concerning the regulation of the activity of the intracellular enzymes have been summarized recently 3. In 1955, DEDtlVE~ proposed that numerous lytic enzymes might be contained in an intracellular entity, the lysosome. An intriguing idea concerning the mechanism of cellular dissolution, resulting in tail disappearance, is that it might be caused by the controlled disintegration of the tail lysosomes. This would cause a release of the Abbreviation: Ta, 3',3,5-triiodothyronine. * Present address: Department of Physiological Chemistry, Medical Science Building, The University of Wisconsin, Madison, Wisc.
Biochim. Biophys. Acta, 93 (1964) 635 643
636
H. KUBLER, E. FRI ED EN
hydrolytic enzymes and the eventual self digestion of the affected cellsa, 5. There is little doubt that as the tail recedes all structural elements disappear and the enzymes claimed to be lysosomal are retained (Table IV). In order to study these tissue changes further, we have examined the activity of the lysosomal enzyme, ~-glucuronidase (/3-D-glucuronide glucuronohydrolase, EC 3.2.I.3I), during spontaneous and induced metamorphosis. A previous report by PRICE AND FRIEDE• 6 established that/3-glucuronidase did not appear to undergo any detectable molecular alteration during metamorphosis. MATERIALS AND METHODS
Animals This study was performed exclusively on Rana grvlio tadpoles collected at different times of the year and from ponds in the Tallahassee, Florida area. They were fed a diet of collard greens and Bacto-Veal and maintained in an aireonditioned laboratory. The animals used in each series of experiments were all of similar size and leg development and from the same source, although a common genetic origin cannot be guaranteed. Induced metamorphosis As described earlier 7, the tadpoles were injected with IO-~/,moles (6. 7/,g of the sodium salt) T 3 per g of tadpole. Controls were injected with amphibian Ringers solution (pH 8.7). The T a was dissolved in Ringers with sufficient sodium hydroxide to dissolve it and all solutions used were freshly prepared. Protein determination The concentration of soluble protein in the Io5ooo x g supernatant was determined by the method of LOWRY et al. 8 using crystalline serum albumin as a standard. [3-Glucuronidase assay The enzyme assay is a modification of the procedure of FISHMANet al. 9 To o.Io ml of a properly diluted sample, 0.80 ml of o.Io M acetate buffer (pH 4.5) and o.Io ml of a O.OLOM phenolphthalein ]3-glucuronide solution were added. After the samples were incubated at 37 ° for 60 rain, 5.0 ml of 0.20 M glycine buffer (pH lO.4) in 0.2 M NaC1 was added. For samples with low activity 2.0 ml of glycine buffer (pH 11.2) was added. This stopped the reaction and produced the red color of the phenolphthalein liberated by the enzyme. Absorbancy was measured at 552 in/, in a Beckman DU spectrophotometer. The enzyme activity is expressed in units per g wet weight of tail tissue. A unit (equivalent to one FISHMAN9 unit) is defined as being equivalent to I / , g of phenolphthalein liberated in I h at pH 4-5 and 37 ° from o.ooi M phenolphthalein fi-glucuronide solution in a total volume of I ml. Specific activity is defined as the ratio of the units of enzyme activity to the protein content of the enzyme solution in mg per g of wet tissue weight. At the wavelength at which the readings were taken, no fading occurred within 15 min. Blanks contained all components of the reaction except the substrate. Under the above conditions the liberation of phenolphthalein is a linear function of time and of enzyme concentration. However, it should be noted that 0.25 M sucrose produces 25-30 % inhibition of enzyme acBiochim. Biophys. ,4cla, 93 (I964) 635-643
LYSOSOMES AND TADPOLE TAIL fl-GLUCURONIDASE
637
tivity 5. Since all assays were performed in o.25 M sucrose, the inhibition is assumed to be constant in all cases and no correction was made.
Preparation of the variousfractions for fl-glueuronidase measurements For the determination of soluble fl-glucuronidase activity of entire tails, six tails of tadpoles or froglets were homogenized in four times their weight of 0.25 M sucrose containing 0.o2 M Tris buffer (pH 7.2). The time of homogenization was 30 sec in a Virtis blendor and I rain in a P o t t e r - E l v e h j e m type homogenizer, equipped with a conical pestle. After differential centrifugation for IO min at 60o × g and 9600 × g and 30 rain at lO5 ooo x g (Spinco Model L Rotor No. 4o), the lO5 ooo × g supernatant was analyzed for the activity of soluble fl-glucuronidase. The activity of soluble fl-glucuronidase was determined on the different parts of the tail from the same animal: the skin, the fin (from which the skin was removed) and the muscular portion. About 7 ° mg of each tissue was homogenized in a small P o t t e r - E l v e h j e m type homogenizer for 30 sec. After differential centrifugation using the swinging bucket rotor, SW 39, specific activity of fl-glucuronidase was determined as described above. For the determination of fl-glucuronidase (soluble plus bound) Triton X - I o o (Robin and Haas Company, Philadelphia, Pa.) was added to the 6o0 × g and 9600 × g supernatants in order to disrupt any intact particles. The residues containing the lysosomal fraction (960o × g residue) and the microsomal fraction (lO5OOO x g residue) were suspended in 0.5 ml 0.25 M sucrose and o.40 ml o.Io N acetate buffer (pH 4-5) in 0.25 M sucrose and o.Io ml 1 % Triton in 0.25 M sucrose. The mixture was incubated in 37 ° for 8 rain, cooled for 2 rain and centrifuged for 30 min at 1o5000 × g in order to release the enzyme. The enzyme activity in the supernatant was measured. The resulting pellet was also tested for enzyme activity to insure a complete release of the enzyme. RESULTS
Soluble fl-glucuronidase activity during spontaneous metamorphosis The increase in the specific activity of soluble fl-glucuronidase during spontaneous metamorphosis is illustrated in Fig. I. Each point represents 3 groups and each group contains 6 tails. The developmental stage of the tadpoles or froglets is usually expressed as the ratio of hindleg length to tail length. Fig. I shows that there is no increase in specific activity at the stage when tadpoles have no legs including the time when the hindlegs and frontlegs are breaking through and there is still considerable growth. At a hindleg to tail ratio in the range o.9-1.1 (initial tail length 65 mm), tail reduction starts and an increase in the specific activity of fl-glucuronidase is observed. At an advanced stage of metamorphosis, hindleg to tail ratio of 12 (final tail length 5 ram), a 34-fold increase in the specific activity of tail fl-glucuronidase is achieved. Since at this stage the tail is I / I I of its original weight, these data suggest that there is a 3-fold increase in the total amount of soluble fl-glucuronidase in the tail. Likewise, the large increase in specific activity cannot be attributed to a decrease in soluble protein. The amount of soluble protein decreases slightly at first and then remains constant (Fig. 4). As tail resorption starts, the fins are absorbed first. Therefore, preparations of Biochim. Biophys. Acta, 93 (1964) 635-643
H. KUBLER, E. FRIEDEN
638
skin, skinless fin and muscle were studied for changes in fl-glucuronidase specific activity in these various parts of the tail. The results (Fig. 2) show that while the initial activities are different, the increase in specific activity of soluble fl-glucuronidase in these three tissues occurs simultaneously with the onset of the tail resorption. SPECIFIC
ACTIVITY
g
OF
"o
"~ 15C
SKIN
zo
IO0i
ili
ID
,7
• *4
I
0
2
,
I
4
,
1
,
I
6 8 LEG/TAIL
l
1 I0
,
I~j__L
I
0.5
12
Fig. I. The increase of specific activity of soluble fl-glucuronidase during s p o n t a n e o u s m e t a m o r phosis. The e n z y m e activity was determined in the s u p e r n a t a n t after centrifugation at 1o5oo0 × g. The horizontal axis refers to the ratio hindleg length: tail length; the vertical axis refers to the specific activity of the enzyme in t e r m s of lO u n i t s of enzyme a c t i v i t y per m g protein per i g of tail (wet).
0.6
....... L___
0.7 0.8 LEG/TAIL
t
1
0.9
1.0
Fig. 2. Tile specific activity of soluble fl-glucuronidase in skinless fin, skin and muscle at the beginning of s p o n t a n e o u s m e t a m o r p h o s i s . Each point represents six individual tails. Coordinates as in Fig. i.
TABLE I ACTIVITY OF
SOLUBLE
fl-GLUCURONIDASE
IN
UNITS
PER
g
OF
TADPOLE
TAIL
A, induced metanmrphosis Hindleg length when injected and temperatures at ~kick the tadpoh's arc kept
0. 5 cm (kept at 26 °) 4.0 cm (kept at 23 _7 2~)
4.5 days:
5.5 days,"
7 days;
40 % tail reduction
48 % tail reduction
72 % tail reduction
i6o-i9o
3oo-33 o --
dead 132o-138o
B, spontaneous metamorphosis H indlcg length and temperatures at which the tadpoles are kept
0. 5 cm (kept at 26 "~) 4.0 cm (kept at 2 3 :: 2 )
4.5 oo tail reduction
930-97 ° -
72 % tail reduction
-18oo-19oo
fi-Glucuronidase activity during induced metamorphosis When metamorphosis is stimulated by Ta, the increase in soluble fl-glucuronidase activity is about i/a of the increase observed for the same amount of tail reduction 13tochim. t3iophys. Acta, 93 (I964) 035 043
639
LYSOSOMES AND TADPOLE TAIL ~-GLUCURONIDASE
occurring during spontaneous differentiation. As shown in Table I, when a later stage is used for induction, the enzyme activity approaches that of the corresponding level of tail reduction in normal metamorphosis. _.J <
I-
z ~ 5o 0 w I1.
/
/
I'--I. . . . .
/v--~---- t
-I
//
m 30 -
m
I I I CONTROL
?--I--~"
_.1 o u~
T3
z
~ 30 rn 0
~,o
i0
I
0
,
I
2
p
I
,
4
DAYS AFTER
I
6 T5
Fig. 3- The increase of soluble protein (supern a t a n t after 10500o x g centrifugation) per g of tail (wet) in induced m e t a m o r p h o s i s .
E
SPONTANEOUS [
L
~0% DECREASE
I
t
,
4o% IN
TAIL
,
I
70% LENGTH
Fig. 4. A c o m p a r i s o n of the a m o u n t of soluble p r o t e i n in the 105000 × g s u p e r n a t a n t in spont a n e o u s a n d induced m e t a m o r p h o s i s as a function of the percent decrease in tail length. A 70% decrease corresponds to a leg: tail ratio of a b o u t 2. 5 .
It was suspected that the large increase in cathepsin (EC 3.4.4.9) activity 1°'2° might result in a solubilization of tail protein. Therefore, we examined the change in the concentration of soluble tail protein during induced metamorphosis. Fig. 3 shows that after T 3 injection a 60 % increase in soluble protein occurs. As shown in Fig. 4 the amount of soluble protein is twice that in spontaneous metamorphosis at the same decrease in tail length. This increase in soluble protein is not dependent on the stage of the tadpole used for injection. Thus, the change in specific activity of tail fi-glucuronidase would be reduced b y a factor of two if expressed on the basis of soluble protein content.
Experiments testing the possible lysosomal nature of tail fl-glucuronidase Since lysosomes are believed to be highly sensitive to rupture by physical forces, tadpole and froglet tails were homogenized in both isotonic and hypotonic medium for varying periods. From the data in Fig. 5 it appears that no additional enzyme is solubilized or released from tadpole tails after 15 sec of treatment in a "Virtis" blendor. If the tail contained an appreciable amount of enzyme not released by homogenization in 15 sec, the appearance of this enzyme in tail supernatants would have been expected. In the froglet, fl-glucuronidase is continuously released for 45 sec. If tail fl-glucuronidase is lysosomal and the lysosomes break down as metamorphosis proceeds, the release of lysosomal bound fi-glucuronidase should increase the amount of soluble fl-glucuronidase during tail resorption, while the total amount of enzyme activity remains constant. To measure the amount of bound activity the enzyme was released from the mitochondrial-lysosomal fraction, the residue at 9600 :.7 g, b y incubation with o.I % Trition X-Ioo. The results summarized in Table II show about a thirty-fold increase of fl-glucuronidase activity in the "particle" bound tYiochim. Biophvs. Acta, 93 (1964) 635-643
640
H. KUBLER,
E.
FRIEDEN
and soluble enzyme per g of tail tissue. The total increase per tail in both fractions is three-fold. Thus, the data indicate that there is no direct transformation of fl-glueuronidase from the particle bound to the soluble state in the autolyzing tail. Q hi
N _J m
=, 0 (/3
FROGLET
30C
_
Z
200
g IOC :3
20
40
SECONDS
60
IN BLENDER
Fig. 5. Tadpole and froglet tails were blended in o. 25 M sucrose or distilled water for varying lengths of time in the "Virtis" blendor. After centrifugation at 1o50oo × g for 3o rain the resulting supernatant was assayed for fl-glucuronidase. TABLE I1 THE
SOLUBLE fl-GLUCURONIDASEACTIVITY OF TADPOLE TAILS DURING SPONTANEOUS METAMORPHOSIS
INCREASE
IN BOUND
AND
Each number represents the average of 4 experiments.
Stage of tadpole
Ratio of lengths hindleg/taiI
Tail weight ( g)
Releasable fl glucuronidase activity from "Lysosomal" fraction (units) One tail
Early Intermediate Late
0.2 o.9 7.0
0.82 o.73 0.073
29 65 84
I.o g of tail
35 89 i ~5o
Soluble fl glucuronidase activitYsupernatantinio 5 ooo × g (units) One tail
r.o g of tail
85 246 226
lO3 337 3092
Intracellular localization of fl-glucuronidase in tadpole tails The intracellular localization of tadpole fl-glucuronidase was studied in more detail. Tail homogenates were subjected to differential centrifugation to yield a nuclear fraction (600 x g residue), a mitochondrial-lysosomal fraction (9600 ; g residue) a n d a m i c r o s o m a l f r a c t i o n (lO 5 ooo ~, g residue). T h e t o t a l a c t i v i t y in t h e r e s p e c t i v e s u p e r n a t a n t s a n d t w o of t h e r e s i d u e s was d e t e r m i n e d a f t e r t r e a t m e n t w i t h o . I o % T r i t o n X - I o o to release b o u n d e n z y m e f r o m all particles. As s h o w n in T a b l e III, t h e a c t i v i t y r e m a i n e d p r e d o m i n a n t l y s o l u b i l i z e d in e a r l y t a d p o l e s w h i c h were at t h e s t a g e p r i o r t o t h e m a j o r i n c r e a s e in t h e specific a c t i v i t y of fi-glucuronidase. A b o u t 20 % of t h e a c t i v i t y s e d i m e n t e d w i t h t h e m i t o c h o n d r i a l - l y s o s o m a l f r a c t i o n a n d I 0 % in t h e m i c r o s o m a l f r a c t i o n . I t was possible to a c c o u n t for t h e d e c r e a s e in t h e a c t i v i t y of e a c h s u p e r n a t a n t r e p o r t e d in T a b l e I I I b y d e t e r m i n i n g t h e r e l e a s a b l e e n z y m e w h i c h a c c o m p a n i e d e a c h residue. F o r e x a m p l e , at an l e g : t a i l r a t i o of 0. 9 t h e a c t i v i t y of t h e s u p e r n a t a n t f r o m Biochim. Biophys. Acta, 93 (I964) 635-643
641
LYSOSOMES A N D T A D P O L E T A I L ~ - G L U C U R O N I D A S E TABLE
III
MAXIMUM RELEASABLE ~-GLUCURONIDASE ACTIVITY OF VARIOUS TAIL FRACTIONS
E a c h number represents IO experiments. Hindleg:tail = 0.2
Hindleg:tail = 0. 9
Hindleg:tail -
x.2-
L3
Supematant after centrifugation at
Units/g
S .D.
Units/g
S.D.
Units/g
S.D.
600 × g 96oo × g lO 5 ooo x g
119 94 81
0. 3 o.3 0. 3
312 253 217
5.4 5.3 5.2
591 485 432
4 .1 4 .o 4.0
the 96oo × g centrifugation had decreased by 59 units and precisely 59 units of enzyme activity were obtained from the Triton treated residue from this fraction. The next fractionation resulted in a decrease of 36 units and 35 units were recovered from the microsomal residue. Satisfactory recoveries of the enzyme in the appropriate residue were obtained in every experiment reported in Table III. DISCUSSION
The large increase in /~-glucuronidase activity establishes this enzyme as one of a group of tail enzymes whose specific activity increases significantly during anuran metamorphosis. As summarized in Table IV, numerous hydrolytic enzymes increase many-fold during the transition of tadpole to frog. Next to the thirty-four-fold increase in /~-glucuronidase reported here, the most impressive activity increase is shown by the intracellular protein hydrolase, cathepsinl°, ~°. A high level of acid phosphatase (EC 3.1.3.2) activity in the regressing Rana pipiens tadpole tail has been demonstrated cytochemically by NOVIKOFFll. We regard it as more than coincidence that all of these enzymes, except collagenase (EC 3.4.4.I9), have been described as lysosomal by DEDUVE 12 and NOVIKOFF13. However, it must be emphasized that several enzymes usually considered lysosomal, e.g. RNAase (EC 2.7.7.16) and lipase TABLE
IV
CHANGES IN ACTIVITIES OF TADPOLE-TAIL ENZYMES DURING METAMORPHOSIS
Several tail e n z y m e s are reported ~ to have no significant increase in a c t i v i t y i n c l u d i n g R N A a s e , lipase, and a protein hydrolase (at p H 8.7). A T P a s e decreases in a c t i v i t y 19 a b o u t 5 I o - f o l d . Relative increase in activity*
Enzyme
fl-Glucuronidase CathepsinX0,20 Collagenase zl Acid phosphatasen,~2, za A protein hydrolase (optimum p H 4.2) 24 Dipeptidases (several kinds)24, 25 D N A a s e ( E C 3 . I . 4 . 5 ) 26
Alkaline phosphatase ( E C 3 . I . 3 . I . ) 2 a
34 22 io 4 IO 9 3-7 5 3
* Relative increase in a c t i v i t y is defined as the ratio of the e n z y m e a c t i v i t y at tile conclusion of spontaneous or induced metamorphosis to the a c t i v i t y found in the early tadpole. Biochim.
B i o p h s , s. A c / a ,
93 (1964) 6 3 5 - 6 4 3
642
H. KUBLER, E. FRIEDEN
(EC 3.I.I.3), do not increase in activity. The possible participation of lysosomal enzymes in the autolysis of the tadpole tails during anuran metamorphosis has been suggested earlier 3,5. However, these data do not yet offer support for the existence of lysosomes in the tadpole tail or the presence of tail fi-glucuronidase in a discrete cell particulate. Assuming that the properties of tadpole tail "lysosomes" are identical with those of rat-liver lysosomes, the "lysosomal" enzymes should be particle-bound and released by detergents or by prolonged homogenization. With tadpole tails, prolonged homogenization did not release particle-bound enzyme, but soluble activity was obtained from froglet tails by this procedure (Fig. 5). It is still possible that the lysosomes are first formed when the tail reduction starts, or that the enzyme is present in an inactive form in tadpole tail "lysosomes" and therefore not detectable, or that the lysosomes are destroyed by the mild homogenization process. No evidence for the appearance of an activator or the loss of an inhibitor was obtained after mixing with the various fractions of supernatants prepared from homogenates of tadpoles and froglet tails. The sum of the activity of the corresponding fractions was always obtained. The amount of activity did not change after subjecting supernatants to dialysis. These results do not exclude the possibility that an activator or an inhibitor might have been destroyed during the preparation of the tail tissue. PRICE AND FRIEDEN6 compared the properties of partially fractionated tadpole and froglet fl-glucuronidase and found no significant differences between these two enzyme preparations. It was observed that there occurs a three-fold increase in total activity per tail in both the particulate "lysosomal" (residue 96oo ~'
LYSOSOMES AND TADPOLE TAIL ~-GLUCURONIDASE
643
enzyme and therefore not the most favorable enzyme to test the lysosome mechanism of tadpole tail autolysis. I t was observed that the change in fl-glucuronidase activity was greater during spontaneous than induced metamorphosis for a corresponding tail reduction. There is ample evidence that T 3 treatment does not always produce an effect identical with those observed during the slower apparently more ordered changes in spontaneous metamorphosis. Strong morphological as well as biochemical differences have been noted earlier 3. Considerable variation has also been observed in enzymic responses. Tail-ATPase activity declines five- to ten-fold during spontaneous metamorphosis, but increases about 1/3 after T3 treatment 17. In m a n y other biochemical responses during metamorphosis such as the increase in serum proteins TM and the changes in hemoglobin m, comparable Ta effects are even more difficult to demonstrate. ACKNOWLEDGEMENT
This research was supported in part by grant C-3oo6 from the U.S. Public Health Service. This is paper No. 19 in a series on the biochemistry of amphibian metamorphosis. REFERENCES 1 C. A. TAYLOR AND J. J. KOLLROS, Anat. Record, 94 (1946) 72 T. P. BENNETT AND E. FRIEDEN, in M. FLORKIN AND H. S. MASON, Comparative Biochemistry, Vol. 4, P a r t B, Academic Press, N e w York, I962, p. 483 . 3 E. FRIEDEN, Principal Biochemical Changes During Amphibian Metamorphosis. Proceedings of the Second International Conference on Congenital Malformations, July, 1963, New York (in the press). 4 C. DEDuvE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS, Biochem. J., 60 (1955) 604. s C. DEDuvE, Funhtionelle und Morphologische Organization der Zelle, \Viss. Konf. der Ges. dtsch. Naturf. u n d Arzte, Springer-Verlag, 1963, p. 209. 6 S. PRICE AND E. FRIEDEN, Comp. Biochem. Physiol., IO (1963) 245. 7 E. FRIEDEN AND G. W. WESTMARK, Science, 133 (1961) 1487. 8 0 . I-1. LOWRY, N. J. ROSEBOROUGH, A. C. FARR AND B. J. RANDALL,J. Biol. Chem., 193 (1951) 265, 9 \¥. H. FISHMAN, B. SPRINGER AND R. BRUNETTI, J. Biol. Chem., 173 (1948) 44910 R. WEBER, Induktion und Morphogenese, 13. Coloq. der Ges. f. Physiol. Chemic, Mosbach, Springer-Verlag, 1962, p. 235. 11 A. B. NOVIKOFF, in D. RUDNICK, Developing Cell Systems and Their Control, Ronald Press, New York, 196o, p. 167. 12 C. DEDUVE, in T. HAYASHI, Subcellular Particles, Ronald Press, New York, 1959, p. 128. 13 A. B. NOVIKOFF, in J. BRACHET AND A. E. MIRSKY, The Cell, Vol, 2, Academic Press, N e w York, 1961, p. 299. 14 K. PAIGEN, Exptl. Cell Res., 25 (1961) 286. 15 N. PASETTO, Inform. Med. Sez. Clin. Sci., 19 (1955) 287. 16 R. WEBER, in A. U. S. DE REUCK AND M. P. CAMERON (Eds.),CibaFound. Syrup. on Lysosomes, J. A. Churchill, L o n d o n , 1963, p. 282. 17 E. FRIEDEN AND H. MATHEVVS, Arch. Biochem. Biophys., 73 (1958) lO7. is A. E. HERNER AND E. FRIEDEN, J. Biol. Chem., 235 (196o) 2845. 19 C. D. TRADER, J. S. \VORTHAM AND E. FRIEDEN, Science, 139 (1963) 918. 2o R. WEBER, Experientia, 13 (I957a) 153. 21 j. GROSS AND C. M. LAPIERE, Proc. Natl. Acad. Sci., 48 (1962) l o l 4. 22 R. WEBER AND B. NIEHUS, Helv. Physiol. Acta, 19 (1961) lo 3. 23 T. YANAGISAWA, Rept. LiberalArts Sci. Fac. Shizuoka Univ. Nat. Sci., 4 (1953) 20; 5 (1954) 3324 E. URBANI, Rend. Ist. Lombardo Sci. Lettere B, 92 (1957) 69. 25 A. M. ZACCHEI, Rieerca Sci. suppl., 24 (1954) 1489. 2~ J. R. COLEMAN, Biochim. Biophys. Acta, 68 (1963) 141.
Bioehim. Biophys. Acta, 93 (1964) 635-643