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Enzyme patterns in isolated mitochondria from embryonic and larval tissues of Xenopus
IlEVELOPhfENTAL
BIOLOGY,
4, 452472
( 1962)
Enzyme Patterns in isolated Mitochondria from Embryonic and Larval Tissues of Xenopus’ R. WEBER AND E. J. BOELL Department
of Zoology,
Yale Uniuersity,
Accepted
December
New
Huoen,
Connecticut
26, 1961
INTRODUCTION
During the past decade and a half, impressive studies have been made on the structural and biochemical characteristics of the mitochondria of fully differentiated cells. These cytoplasmic particles are of interest not only because of their elaborate morphological organization, but also because they serve as important centers of metabolic activity. They contain, among others, the constellation of enzymes concerned with oxidative phosphorylation (Schneider and Hogeboom, 1951; Lindberg and Ernster, 1954; Ernster and Lindberg, 1958; Novikoff, 1961) , and they are, therefore, important agents in energy transfer in cells and tissues. Furthermore, it has been demonstrated that the mitochondria derived from a given adult tissue comprise a heterogeneous population of cytoplasmic constituents (de Duve et al., 1953; Novikoff et al., 1953; Kuff et al., 1956; Thomson and Moss, 1956). And there is some evidence to suggest that mitochondria obtained from different tissues have specific biochemical characteristics (Felix et al., 1954; Green and Beinert, 1955). It has been shown, in a number of now classical studies (Wilson, 1928) and more recently through the use of the electron microscope, that mitochondria are important inclusions of eggs and embryonic cells (Weber and Boell, 1955; Weber, 1956; 1958; Eakin and Lehmann, 1957; Rebhun, 1956; Karasaki, 1959) and that specific enzymes are associated with them (Cleland, 1951; Gustafson, 1952; Yeas, 1954; Boell and Weber, 1955; Solomon, 1959). ’ This work was supported by grants from the National Science Foundation Na(G-4288 and G-8771 ) and, in part, by a g rant from the Schweizerischen tional fonds. 452
ENZYME
I’ATTERXS
IX
hlITOCHONDRIA
453
From time to time, attention has been focused on the developmental significance of mitochondria, with special emphasis on such questions as variations in their relative number, their distribution within the cell or within various presumptive regions of the embryo, developmental changes in their cytochemical and biochemical properties, changes in morphology, et cetera (Gustafson, 1959; Boell and Weber, 1955; Eakin and Lehmann, 1957; Reverberi, 1957; Kaye, 1958; Pasteels, 1958; Shen, 1958; Novikoff, 1961). Although the literature on the subject is voluminous, little or no concrete evidence exists which can help us to determine whether the specific role of mitochondria in embryonic cells is fundamentally any different from that in mature cells. As Novikoff (1961, page 367) has recently stated, “The precise role of mitochondria in embryonic development remains to be established. Apparently, mitochondria of ova and embryos possess the same fine structure as in adult cells . . . but changes may occur during development. There is little reason to doubt that most possess essentially the same oxidative, phosphorylating, and other activities as found in mitochondria of mature tissues. . . . The unanswered question is whether they serve exclusively as suppliers of energy and metabolites, or have additional, more specific directive roles in development.” In view of the results summarized above, some of the changes in enzyme activity known to occur during embryonic development (Boell, 1955; Shen, 1955; Moog, 1958; Duspiva, 1955) may reasonably be expected to reflect ontogenetic changes in the properties of mitochondria. The present study was undertaken to explore this possibility. In this approach, emphasis has been shifted from mitochondria to mitochondria as objects of as agents of embryonic differentiation embryonic differentiation. Our preoccupation with the latter aspect of the subject does not imply, however, that we discount the possible importance of mitochondria in the former. Indeed, any cellular constituent concerned directly or indirectly with protein synthesis must have significance in the differentiation process. In previous work we have been able to show a progressive increase in cytochrome oxidase activity in isolated mitochondria from embryonic and prefeeding stages of Xenopus (Boell and Weber, 1955; Weber and Boell, 1955). This was interpreted as the result of differential growth rates of the members of a heterogeneous population of mitochondria as well as intrinsic changes in individual mite-
454
WISER
AND
BOELL
chondria. The results of recent observations of amphibian embryonic cells with the electron microscope are consonant with this view for they show that the mitochondria undergo progressive differentiation both in shape and in internal fine structure (Eakin and Lehmann, 1957; Karasaki, 1959). These findings have led us to extend our earlier experiments on the biochemical changes in mitochondria during embryonic differentiation of Xenopus. Our previous study involved only a determination of cytochrome oxidase activity in mitochondria derived from embryos of different ages. In the present work, comparative data are presented on the specific activities of four enzymes (cytochrome oxidase, ATPase, acid phosphatase’ and cathepsin) in mitochondria obtained from undifferentiated tissue primordia and from fully differentiated larval tissues. We shall try to answer the following questions: (1) Are mitochondria of undifferentiated tissue primordia characterized by specific enzyme patterns? (2) Do tissue-specific changes occur in mitochondrial enzyme patterns during differentiation? MATERIAL
Preparations
of Mitochondriul
AND
METHODS
Fractions
Mitochondria were isolated by differential centrifugation either from whole and dissected tailbud embryos or from tissues of full grown larvae of Xerwpus laevis. 3,4 An important point in our experimental design was the investigation of mitochondria from corresponding tissues at an early stage of development and at the completion of morphological and physiological differentiation. As a source of relatively undifferentiated tissue, tailbud embryos (stage 33/34, Nieuwkoop and Faber, 1956) were selected. These ‘We do not know whether or not in embryonic tissues acid phosphatase is associated with specialized cell particulates such as those derived by de Duve (1957) from adult tissue and characterized by him as “lysosomes.” If it is, we were unable to clear our mitochondrial preparations of lysosomes, for they always contained acid phosphatasc in high concentration even after repeated washing and recentrifugation, cf. page 462. ‘We are greatly indebted to Professor F. E. Lehmann, Zoological Institute of the University of Bern, Switzerland, who kindly supplied us with adult Xenopus. ‘We also wish to express our gratitude to the “Ciba” A-G Basle, Switzerland, for a sample of choriogonadotropic hormone which never failed to induce spawning.
ENZYME
PATTERNS
were placed in amphibian Ringer membranes were removed. The and the remaining body portion into a dorsal and a ventral half
IN
MITOCHONDRIX
455
solution, and the jelly and vitelline heads were cut off and discarded, of each embryo was then dissected (Fig. 1). By this procedure it was
DORSAL HALF = “MESODERM” I
I
\
“ENTRLL
HALF = “ENDODERM”
FIG. 1. Dissection of taiibud embryos. embryos were cut into dorsal and ventral “mesoderm” and “endoderm” cells.
The broken lines indicate how the halves which served as sources for
possible to collect in a relatively short time fragments of embryonic tissue which were predominantly mesodermal (dorsal halves) or endodermal (ventral halves). These fragments, or in some instances whole embryos of the same stage, were transferred to ice-cold 0.28 M sucrose + 10e3M Versene, neutralized to pH 7.2 with NaOH ( = S-Vmedium). As samples of differentiated tissue, derived from endoderm and mesoderm, respectively, liver and tail muscle of Xenopus larvae at the beginning of metamorphosis (stage 56/57, Nieuwkoop and Faber, 1956) were taken and, after transfer to chilled S-V-medium, were freed from contaminating tissues. Homogenates were prepared at low temperature by grinding tissue samples in a conical all-glass microhomogenizer with a small amount of S-V-medium. The tissue brei was then further diluted with S-Vmedium, giving concentrations of 10 dorsal or ventral fragments per 100 ~1 homogenate and 10 mg liver or tail tissue per 100 ~1 homogenate. Since our main interest lay in the biochemical properties of mitochondria, we did not attempt any complicated fractionation pro-
456
WEBEH
AND BOELL
cedure, but followed our earlier method which with some minor modifications was found satisfactory. The homogenates were fractionated by means of a microcentrifuge with a head accommodating 12 horizontally rotating tubes, each tube measuring 28 mm in length and having a capacity of 0.5 ml. In order to reduce heating effects, due to high speed centrifugation, the centrifuge was operated in a refrigerator at a temperature of O-5” C. This measure was effective, except for runs at top speed during which the tubes were slightly heated. In these cases, however, exposure to slightly elevated temperature lasted a few minutes only and hence could not be too deleterious to the mitochondrial preparations. Table 1 shows a survey of the isolation procedure by which relaTABLE I FRACTIONATION PROCEDURE
300 ~1 in 0%
Homogenate :I[ sucrose + 1O-3 !lI Versene 10 min . . 700 q 1
1
It, : Yolk, pigment granules, tissue debris + 300 ~1 S-V-medium I I 10 min . TOO q 1 SW RIW + 300 ~1 S-V-medium I 10 min . .
iO0 q
I SW RIW ’ + 150 ~1 S-V-medium = Fraction 1: Yolk, pigment granules, tissue debris * Supernatants
SW + SW’ I I I 1* I* R2h R,, ---_--
Sl I 1 15 min
. . 17,500 g
A Ran 2
k? s I + 300 ~1 S-V-medium / I I 1 15 min . . . 17,500 g /
1* /\ l&w Rz L_-+ ca. 150 ~1 S-V-medium
S, Final supernatan,i including microsomes
= Fraction 2: Mitochondria, some pigment granules
from these washings were discarded.
‘The microcentrifuge was designed Hohnevej, Brendby Strand, Denmark.
and
constructed
by Mr.
Ole
Dich,
18
ENZYME
PATTERh-S
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457
tively pure mitochondrial preparations could be obtained in a rather short time. The volumes of the homogenate were measured by means of constriction pipettes and those of the fractions were determined by weighing. The data of the distribution of total nitrogen showed that in spite of the small amounts of starting material excellent recoveries and well reproducible values were obtained, thus indicating only slight losses of material during differential centrifugation. Enzyme
Assays
The activities of cytochrome oxidase, ATPase, acid phosphatase, and cathepsin were determined by means of micromethods which will be described in detail below. For each enzyme studied, a series of experiments was performed on the assay technique alone in order to make sure that determinations of enzyme activity were done under optimal conditions. In some experiments, the enzyme activities of all fractions including the washings were determined. In most cases, however, the activities were determined only on homogenates and isolated mitochondria. It should be noted that the relative activities of enzymes in mitochondrial preparations were found to vary somewhat from experiment to experiment, probably because of biological variability in the embryos obtained from different females. Cytochrome oxidase activity was determined at 25” C by means of the Cartesian diver technique, using siliconed divers of approximately 10 ~1 total volume. The diver filling consisted of: 0.5 ~1 l/10 N NaOH ( bottom drop), 0.4 ~1 substrate (side drop, containing 0.1 M p-phenylenediamine and 2.5 x lo-” M cytochrome c in 0.1 M phosphate buffer of pH 7.4), 0.6 ,~l homogenate or mitochondrial suspension (neck seal), 0.7 ,J paraffin oil, and mouth seals measuring about 3 mm in length. After a period of temperature equilibration, usually 10-20 minutes, enzyme and substrate were mixed by raising the pressure in the diver system, and the reaction was then followed for a l-hour period. The readings were taken at intervals of 5-10 minutes, and in most cases the oxygen uptake was at a linear rate, at least during the first 30 minutes. The concentration of the enzyme samples was adjusted so that the pressure change in the divers ranged between 60 and 120 mm per hour. Final readings were corrected for substrate autoxidation, but, as was true in our previous work, endogenous respiration of homogenates and of mitochondrial suspensions was negligible.
458
WEBER
AND
BOELL
Mitochondrial ATPase activity was assayed, following in part the methods of Kielley (1953, 1955) and Monroy (1957). To 5 ,J of substrate ( ATP-disodium-salt” 1.5 X lo-” M + MgCl, 1.5 X 10e3M barbital buffer 0.1 M pH 7.6), 5 ~1 of the enzyme sample was added and incubated for 30 minutes at 25” C. The reaction was stopped by adding 10 ~1 of chilled 5% trichloroacetic acid + 0.5% uranium sulfate (Krugelis, 1950). The tubes were immediately put into a refrigerator at 0” C, in order to avoid acid hydrolysis of ATP. As controls, substrate samples were precipitated before addition of the enzyme aliquots. Thus a correction was made only for free PO,--- present in the enzyme samples, since it was found that, by working at low temperature, spontaneous breakdown of ATP in presence of TCA was negligible. The liberated PO,--- was estimated according to the sensitive calorimetric method of Kuttner and Cohen (1927). The chilled tubes were centrifuged for 5 minutes at 17,500 g and then 10 ~1 of the supernatant was removed. To this aliquot, 10 ~1 each of 4 N HZSOI, 3% ammonium molybdate, and 0.8% &Cl, were added in the order stated, with thorough mixing of the content of the tubes after each addition. The blue color was allowed to develop for 30 minutes and then readings were taken on samples of 20 ~1 at 640-700 rnp (Klett Filter No. 66) by using a microcolorimeter similar to the design of Krugelis (1950). In a series of test readings our apparatus showed an excellent agreement with values obtained from the Beckman DU spectrophotometer at 670 mp. It should be mentioned that the PO,-reaction is extremely sensitive to impurities, which lead to considerable differences in color intensity between duplicate samples. This difficulty can be overcome, however, by a careful cleaning procedure of the tubes, in which they are first immersed in hot cleaning solution (sulfuric acid-sodium dichromate) then rinsed at least five times with glass-distilled water. Bv this means it is possible to reduce variations among triplicates to less than 5%. ATPase activity was expressed as micromoles P X 10m3liberated per microgram TN in 30 minutes (specific activity) and as micromoles P X lo-” liberated per 30 minutes (total activity). Acid phosphatase activity. To 5 ~1 of substrate (3.1 X 1O-2M ‘Obtained
frown the Sigma Co., St. Louis, Missouri.
ENZYME
PATTERNS
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459
sodium-/&glycerophosphate’ + 0.06 211acetate buffer pH = LO), 5 ~1 of the enzyme sample was added, and, after mixing, the tubes were incubated at 39” C for 4 hours. The reaction was stopped by addition of 10 ~1 of chilled 5% trichloroacetic acid (TCA) + 0.5% uranium sulfate and then the tubes were put into a refrigerator at 0” C. AS controls, substrate aliquots were mixed with chilled TCA and to these tubes the enzyme samples were added. Since it was found that in the presence of TCA neither substrate nor homogenate samples released appreciable amounts of P04m--, only a correction for endogenous free PO,--- was made. The calorimetric determination PO,--- was the same as that described for ATPase assays. The activity of acid phosphatase was expressed as micromoles P X lo-” released per microgram TN per 1 hour (specific activity) and as micromoles P x 10m3released per 1 hour (total activity ). C&epic activity was determined according to the calorimetric method of Duspiva (1939) with a mixture of 2% casein “Hammarsten” as substrate in 48% urea and citrate-phosphate-ammonia buffer pH = 4.80. The reaction mixture which consisted of 10 ~1 of substrate and of 5 ~1 enzyme aliquots was incubated for 3 hours at 37” C. The reaction was stopped by adding 100 ~1 of chilled 5% TCA, and then the tubes were left standing for at least 4 hours in the refrigerator. The controls were set up by adding the enzyme to the tubes which contained substrate and TCA samples. For the calorimetric determinations the precipitated reaction mixtures were centrifuged for 5 minutes at 17,500 g; 50 PI of the supernatant was thoroughly mixed with 50 ~1 of 13.5% Na,CO, (anhydrous), and after addition of 50 ,.J Folin-Ciocalteu-reagent (diluted 1:5), the blue color was allowed to develop for 30 minutes. Again, the calorimetric readings were taken on 20-J aliquots by means of the microcolorimeter using a Klett-Filter No. 69 with a spectral range of 660-740 rn&, The enzyme activities were compared to a standard activity curve, obtained from a dilution series of a liver homogenate in S-V-medium. This curve showed a linear relationship between activity and homogenate concentration up to 0.5 mg liver tissue per 100 J homogenate. By means of a standard curve the E values were converted into micrograms casein per 10 ~1 and the activity units for cathepsin were ’ Obtained from the Sigma Co., St. Louis, Missouri.
460
WEBER
AND
BOELL
then expressed as micrograms casein split per microgram TN per hour (specific activity) and as micrograms casein split per hour (total activity ) . All determinations were made in duplicate; the variation between them was found to be less than 5%. Total nitrogen ( TN) was determined by means of an ultramicroKjeldahl method (Boell and Shen, 1954) on duplicate samples that contained 1-15 pg TN. Electron
Microscopy
In order to obtain complementary information on the morphology of the mitochondria that were studied biochemically, samples of all the tissues which served as sources for particulate fractions were observed by means of a Siemens “Elmiskop l”* working at 60 kv and 5 pamp. RESULTS
The Yield of Enzyme Activity
from
Mitochondrial
Preparations
By comparing specific enzyme activities in mitochondrial preparations and in corresponding homogenates, we obtained activity ratios indicating the extent to which the enzymes are concentrated in the mitochondria. The data, summarized in Table 2, show that the acTABLE
2
CONCENTRATION OF ENZYMES IN MITOCHONDRIA (WHOLE HOMOGENATE = l)a ATPave
Whole embryos (gastrula-tailbud) “Mesoderm” halves (tailbud) “Endoderm” halves (tailbud) Liver (larval stage 55) Tail muscle (larval stage 55)
8.2 7.0 12.0 2.9 8.2
Mammalian liver tissue (from Schneider, 1953)
3.1
a (l), (2), (3), (4) = respectively,
(4) (3) (3) (3)
7.1 5.2 6.2 3.1
(4) (3) (3) (1)
(1)
3.9
(1)
1 experiment
2.4
Acid phosphatase
4.2 3.5 3.8 1.5 6.7 1.3
(3) (3) (3) (1) (1)
Cathepsin
4.5 3.8 6.0 2.0 6.2
(4) (3) (3) (2) (1)
2.6
or means of 2, 3, or 4 experiments.
*We are greatly indebted to the Electron Microscopy Division of the Institute of Inorganic Chemistry at the University of Bern for assistance in operating the electron microscope.
ENZYME
PA’ITERNS
IN
MITOCHONDRIA
461
tivities of all of the enzymes studied are considerably greater in mitochondria than in homogenates. The table shows the further extremely interesting point that the degree to which the enzymes are concentrated is different for each of the four enzymes tested and also differs for each of the tissues from which the mitochondria were derived. It seems clear, therefore, that mitochondria from one embryonic tissue cannot be regarded as equivalent to those from another. The data suggest that the pattern of enzyme concentration, as well as the relative activities of the four enzymes studied (Table 3), comprise a set of biochemical characteristics specific for each kind appealing of embryonic or adult tissue. Such an idea is particularly when the remarkable similarity in the enzyme activities of Xen~pus liver and mammalian liver are noted (Table 2). Although the data are not numerous, the similarity in the activity ratios seems to be altogether too close to be purely fortuitous. It was not our intention to make an elaborate analysis of enzyme distribution in the various fractions obtained through differential centrifugation of tissue homogenates. A few measurements of this kind were made, however, so that the total activity of homogenates which could be accounted for by the activity of the mitochondrial fractions could be determined. In general, the relative recovery of enzyme activity in mitochondria in our experiments agreed well with data reported in the literature ( Schneider, 1953)) although absolute recovery was occasionally somewhat lower. This is undoubtedly due to minute losses of mitochondria during the isolation procedure. It may be recalled that, in comparison with the usual techniques for isolating mitochondria, we were working with extremely small volumes of homogenate and particulate suspensions ( microliters instead of milliliters ) . Small losses, insignificant in the usual macro techniques, would thus have a considerable effect on recovery of enzyme activity. In the case of cytochrome oxidase, no appreciable amount of enzyme was ever found in nonmitochondrial fractions. Yolk and tissue debris (R,), the final supernatant (S,), or the washings never showed more than 6% of the activity of the original homogenate. On the other hand, in the case of ATPase, acid phosphatase, and cathepsin, an appreciable percentage of the total activity of the tissue was recovered in the final supernatant. The recoveries in the supernatant on the average amounted to 20% for ATPase, whereas for acid phos-
462
WEBEH
.&SD
UOELL
phatase and cathepsin occasionally as much as 50% could be recovered in the supernatant. This finding is of interest, for on a quite different material, viz., mammalian liver, sedimentation behavior for particles that carry cytochrome oxidase was discovered to be different from that of particles to which acid phosphatase and cathepsin are bound (de Duve et al., 1953). Whether this also applies to particles derived from embryonic and differentiated cells of Xenopus is not known at present. Recent findings on the development of glutamic dehydrogenase activity in differentiating liver tissue of chick embryos, after the 12th day of incubation, indicate inversely changing percentage activity in mitochondria and in the supernatant (Solomon, 1959). To what extent ATPase, acid phosphatase, and cathepsin, found in differentiating tissues of Xen,opus, reflect a similar phenomenon cannot be decided from our data. Cytochrome Oxidase Activity and Differentiated Cells
in Mitochondria
from Tissue Primordiu
The results on cytochrome oxidase will be considered first, since they bear directly upon our main problem, namely the biochemical heterogeneity of mitochondrial populations. In Fig. 2 a comparison is made of the cytochrome oxidase activity per unit of TN for mitochondrial fractions obtained from whole embryos, dorsal halves ( = “mesoderm”) and ventral halves ( = “endoderm”) of the tailbud stage. It may be noted that “mesoderm” mitochondria contain 3.25 times more activity per unit nitrogen than do mitochondria isolated from “endoderm”; the mitochondria from whole embryos show an intermediate level of activity. Now if “mesoderm” and “endoderm” are compared with their derivatives, namely larval tail muscle and liver, one is struck by the fact, that the specific activities of these mitochondria are almost identical with those of the corresponding tissue primordia. Thus the activity ratio for tail muscle:liver amounts to 3.1:I. One may conclude that during development mitochondrial populations from relatively undifferentiated cells ( tissue primordia ) may already show differences in their enzyme content. This result has an important implication which bears upon the fractionation procedure. In view of the different properties of the it is unlikely that equal homogenates which were fractionated, amounts of contaminating “inert proteins” were present in the mito-
ENZYME
PATTE:RIVS
SPECIFIC ACTIVITY
IN
463
MITOCHONDRIA
3.25
2500 Moo 1500 1000 500 0
I
1
EMBRYO
WHOLE EMBRYO \ ‘\
EMBRYO “ENDODERM” FIG. 2. Cytochrome and differentiated larval pg TN.
TAIL MUSCLE
“MESODERM”
LIVER
oxidase in mitochondria of undifferentiated embryonic tissue. The units of specific activity are 10e3 ~1 Oz/hr/
chondria from embryonic tissues (“mesoderm” or “endoderm”) and from differentiated tissues (tail muscle or liver) and that such contamination was responsible for the figures reported in Figs. 2 and 3. Instead, it seems reasonable to regard the observed differences in specific activity as true biochemical characteristics of these mitochondrial preparations. Patterns of Enzyme Activity
in Mitochondriu
of Various
Tissues
Table 3 presents average figures for the specific activity levels cytochrome oxidase, ATPase, acid phosphatase, and cathepsin mitochondria from the various embryonic and larval tissues used this study, i.e., from whole embryos, endoderm, and mesoderm stages 33/34 and from liver and muscle dissected from larvae stages 56/57,
of in in at at
464
WEBER
AA-D
BOELL
TABLE 3 SPECIFIC ACTIVITY~ OF ENZYMES IN MITOCHONDRIA FROM EMBRYONIC AND LARVAL TISKES OF SENOPIJ~ Cytochrome oxidase
Acid
ATPase
phosphataue
CathepGn
Q
R
Q
R
Q
R
Q
R
Tailbud embryo Dorsal half “mesoderm” Ventral half “endoderm”
1038
1.0
4.55
1.0
0.70
1.0
7.18
1.0
2493
2.4
8.86
1.9
2.38
3.4
10.56
1.5
768
0.7
5.52
1.2
0.76
1.1
5.22
0.7
Tail muscle
2030
2.0
18.5
4.1
0.72
1.0
7.07
1.0
649
0.6
32.3
7.1
Liver
10.5
15.0
35.8
5.0
a Q = specific activity; R = ratio of activity compared with tailbud embryo. For description of activity units, see Methods.
The table shows that the specific activities of all the enzymes tested are different from tissue to tissue. It is also apparent that developmental changes in enzyme activity within the mitochondria of a given tissue may involve ( 1) no significant alteration in level, (2) an increase, or (3) a decrease in activity. Furthermore, the figures show that during development the variations in activity of a particular enzyme within the mitochondria of a given tissue are completely independent of those of any of the other enzymes, both in terms of direction and magnitude of change. With cytochrome oxidase, for example, activity declines approximately 15% during development in both liver and muscle. ATPase activity increases in both tissues, but in muscle it does so by only a little more than twofold, while in the differentiation of liver, activity rises by almost sixfold. By contrast, acid phosphatase declines during differentiation of muscle, but during the same developmental period it increases very considerably (fourteenfold) in the liver. The developmental change in catheptic activity is similar in direction to that of acid phosphatase, decreasing in muscle and increasing in the liver, but the extent of change of cathepsin activity is quantitatively quite different from that of acid phosphatase. From a consideration of such data, it appears that the development of a specific pattern of enzyme activity is typical of the mitochondria from each of the tissues studied. The distinctive character of the
ENZYME
PATTERNS
IN MITOCHONDRM
465
changes in pattern of enzyme activity, occurring during morphological and functional differentiation of muscle and liver, are illustrated in Fig. 3. 20-
/ /V,
IO-= 51 -
-
0' /'LIVER /I
. .
-0
ENDODERM
TAIL MUSCLE MESODERM
FIG. 3. Enzyme patterns in mitochondria of undifferentiated embryonic and differentiated larval tissues. The activity levels are plotted in a logarithmic scale which indicates units of specific activity. The figures for ATPase, acid phosphatase, and cathepsin are multiplied by 100 to bring them into convenient scale.
DISCUSSION
In tracing changes in the enzyme activity of mitochondria, during the development of a tissue from its primary germ layer antecedent to the fully differentiated state, it would be desirable to make measurements not only on stages at the beginning and at the end of the developmental period under consideration, but also on a number of intermediate ones as well. Unfortunately, this could not be done in the present study owing largely to still unresolved difficulties in isolating and fractionating the extremely small bits of tissue which embryonic tissues and organs represent, especially in early stages
466
WEBEH
AND
BOELL
Hence, the present study must be regarded as somewhat exploratory rather than comprehensive. But in spite of this, several features in the results obtained stand out clearly and are worthy of further comment. First, it should be emphasized that mitochondria from different tissues, even from the earliest embryos tested, have distinctive characteristics which are revealed in significant quantitative differences in specific activity of particular enzymes. This is shown graphically in Fig. 2 for cytochrome oxidase and in Table 3 for the other enzymes tested. A further point of interest is the fact that the “developmental behavior” of each of the enzymes appears to be unique for a given tissue. This is best seen in comparisons of the activity of a particular enzyme in mitochondrial suspensions from the early whole embryo, from a tissue primordium, and from the differentiated derivative of the primordium (Table 3). For example, the ratio of cytochrome oxidase activity in whole embryo, mesoderm, and tail muscle 1:2.4:2. By contrast, for ATPase activity from the same three sources, the ratio is 1:1.9:4.1. The ratio of cytochrome oxidase activity in early whole embryo, endoderm, and liver is 1:0.7:0.6; for ATPase, it is 1:1.2:7.1; and for acid phosphatase it is 1:1.1:15. These calculations provide the most powerful argument we know against the suggestion that quantitative differences in specific activity, such as those noted, are due to variations in the degree of contamination of the mitochondrial preparation with nonenzymatic constituents. The observed difference in the specific activities of mitochondria from various tissues helps to answer a question which we raised in our earlier study of cytochrome oxidase activity in mitochondria obtained from whole Xenopus embryos (Boell and Weber, 1955). In commenting on the rise in cytochrome oxidase activity per unit of mitochondrial nitrogen which occurs during development, we stated: “The change in the Q value of mitochondria might represent something as simple as the gradual disappearance during development of a contaminant of the mitochondrial fraction. It might also reflect a shift in the ‘population statistics’ of the mitochondrial fraction representing changes in the relative numbers of specific kinds of mitochondria brought about by differential growth of the various tissues of the embryo. On the other hand, it could represent an intrinsic
ESZYME
PATTERNS
IN
MITOCHONDRIA
467
change in the individual mitochondria characterized by the transformation of catalytically inert protein into cytochrome oxidase.” From the discussion above, it seems to us very unlikely that the presence and gradual disappearance of a contaminant in the mitochondrial preparations could in any way account for the changes in specific activity of enzymes reported in this paper. Furthermore, although the possibility of intrinsic changes occurring in mitochondria during development is by no means ruled out, nor is it even lessened, it is now clear that the development of a heterogeneous population of mitochondria must account, to some extent-perhaps to a considerable extent-for the facts observed in the work referred to above. Mitochondria from mesoderm and muscle tissue, as can be seen from Fig. 2, have a threefold greater specific activity of cytochrome oxidase than mitochondria from endodermal tissue. Hence, the progressive increase in the proportion of muscle in the embryo as development proceeds would be expected to lead to a rise in specific activity of mitochondria from homogenates of the whole embryo. Relatively more of the particulates in such a suspension would represent mitochondria contributed by muscle tissue. Any attempt to evaluate the significance of changing patterns in enzyme activity during development leads one to ask whether the observed results are due simply to increases or decreases in the number of units of enzyme in a particular tissue or whether enzyme molecules themselves are undergoing change as development proceeds.” Usually the dynamic properties of enzymes are defined by the Michaelis-Menten constant, K,,,, for a given substrate. We applied this method (\R’eber, unpublished) in an attempt to assess the properties of acid phosphatase which, as will be seen in Table 3, undergoes the most impressive developmental change in activity of the four enzymes tested. Although this work is still in somewhat preliminary form, the main result is summarized in Table 4 and shows that K, is almost constant for mitochondria from embryonic and differentiated tissues (compare Moog, 1961). Many factors, especially the purity of the enzyme preparation, can affect the Michaelis-Menten constant, but, if the results summarized in Table 4 are taken at face value, there seems to be no change in ‘This view has been advanced sion of Moog’s paper ( 1958).
by de Villafranca,
among others,
in a discus-
468
WERER AND
BOPLI.
TABLE 4 MICHAELIS-MENTEX
COSSTAKT AT DIFFERENT
Source of enzyme
(Km)
extract
Whole tailbud embryo Larval liver Larval tail muscle
OF ACID
STAGES
PHOSPHATASE
Km x 10-z * &I<.,,
3.10
3.39 3.80
FROM TISSUES
OF DEVELOP.MENT ,V”
0.81 0.38 0.33
3
14 G
a S.E. = standard error. b N = number of determinations.
affinity of acid phosphatase for its substrate. It would seem, therefore, that the developmental change in acid phosphatase activity is due to an increase in the number of enzyme molecules rather than to changes in the enzyme, as such. In view of the striking differences which have been demonstrated in the enzyme activity of mitochondria from mesoderm and muscle, on the one hand, and endoderm and liver, on the other, the question arises whether any correlation exists between the biochemical findings and the structural organization of these cytoplasmic constituents. It was shown for rat liver mitochondria that cytochrome oxidase, and certain other enzymes, are firmly bound to mitochondrial structure (Hogeboom and Schneider, 1950). More recently, Siekevitz and Watson (1956) have reported that when mitochondria are disrupted oxidase and succinoxidase remain by deoxycholate, cytochrome firmly attached to a fraction identified as consisting of mitochondrial membranes. In view of these observations, it is of interest to note that preliminary results of an electron microscopic study of Xenop~ larval tissues reveal a difference in the fine structure of mitochondria from tail muscle and from liver. In mitochondria from tail muscle, there is an elaborate system of internal cristae with relatively little space for matrix. In liver mitochondria, on the other hand, the cristae are much less numerous and seem to be confined to the periphery, thus leaving a large volume of mitochondrial ground substance or matrix. Similar differences in fine structure have been shown to exist in mitochondria of striated muscle and the parenchymatous cells of the liver in the adult rat (Palade, 1953). It appears, therefore, that the quantitative differences in specific activity of cytochrome oxidase are correlated with morphological differences in the mitochondria. In
ENZYhlE
PATTERNS
IN
h’flTOCHONDHIA
469
view of this, it would be particularly interesting to examine mitochondrial fine structure in a tissue in which cytochrome oxidase and succinoxidase activity increase considerably during development. If enzyme content is positively correlated with the quantity or degree of elaboration of cristae in mitochondria, ontogenetic changes in mitochondrial fine structure might reasonably be expected. It is difficult to assess the developmental significance of the patterns of enzyme activity in mitochondria from embryonic tissues. We do not believe that they are merely fortuitous. At the very least, they constitute biochemical indexes of the functional differentiation of embryonic tissues. They must, furthermore, represent adaptations to their specific physiological requirements. Perhaps they are intimately concerned with developmental processes, but exactly how they may be involved remains a matter for speculation. In any case, further investigation of the specific enzymatic capital of developing tissues and of the fundamental factors which control the synthesis of enzymes and which regulate their structural organization within the cell will continue to provide a challenging and fruitful field of inquiry.
The specific activities of cytochrome oxidase, ATPase, acid phosphatase, and cathepsin in homogenates of embryonic and larval tissues of Xenopus and in mitochondria derived from them have been determined. In all cases, determinations of enzyme activity were made on homogenates and mitochondria from early whole embryos, from tissue primordia, and from the fully differentiated end products of the primordia. The mitochondria from each embryonic or larval tissue studied have distinctive characteristics which appear as significant quantitative differences in specific activity of each of the four enzymes tested. During development, the changes in activity of each enzyme in the mitochondria from a given tissue follow an individual pattern which is independent of the pattern of other enzymes in the same mitochondria. In the case of cytochrome oxidase, a correlation has been found between the fine structure of the mitochondria and the level of enzymatic activity. Liver mitochondria have few cristae and relatively low specific activity of cytochrome oxidase; muscle mitochondria have numerous cristae and a high specific activity of cytochrome oxidase.
REFERENCES BOELL, E. J. (1955). Energy exchange and enzyme development during enrbryogenesis. In “Analysis of Development” (B. H. Willier, P. A. W’eiss, and V. Hamburger, eds.), pp. 520-555. Saunders, Philadelphia and London. BOELL, E. J., and SHEN, S. C. (1954). An improved ultramicro-Kjeldahl technique. Exptl. Cell Research 7, 147-152. BOELL, E. J., and WEBER, R. ( 1955). Cytochrome oxidase activity in mitochondria during Amphibian development. Exptl. Cell Reseurch 9, 559567. &ELAND, K. 1%‘. ( 1951). The enzymatic architecture of the unfertilized oyster egg. Australian J. Exptl. Biol. Med. Sci. 19, 35-45. DE DUVE, C. ( 1957). The enzymatic heterogeneity of cell fractions isolated by differential centrifugation. Symposia Sot. Exptl. Biol. No. X, 50-61. DE DWE, C., GIANETTO, R., APPELMANS, F., and WA~IAUX, R. (1953). Enzymic content of the mitochondria fraction. Nature 172. 1143. DUSPIVA, F. ( 1939). Beitrage zur Histophysiologie des Insektendarmes. I. Untersuchungen iiber die Verteilung der proteolytischen Fermente sowie der Sekretund Resorptionszellen im Darm von Dytiscus marginalis. Protoplasma 32, 211250.
DUSPIVA, F. ( 1955). Zur Biochemie der normalen Wirbeltierentwicklung. Naturwissenschaften 42, 305-311. EAKIN, R. M., and LEHMANN, F. E. (1957). An electron microscopic study of Roux’ Arch. Entwicklungsmech. developing Amphibian ectoderm. Wilhelm Organ. 150, 177-198. ERNSTER,L., and LINDBERG, 0. ( 1958). Animal mitochondria. Ann. Rev. Physiol. 20, 13-42.
FELIX, K., DECKER, M., and ROKA, L. (1954). Dehydrogenase-Aktivitat der Mitochondrien verschiedener Organe. 2. physiol. Chem. Hoppe-Seyler’s 294, 79-85. GREEN, D. E., and BEINERT, H. (1955). Biological oxidations. Ann. Rev. Biothem. 24, 144. GUSTAFSON, T. ( 1952). “Nitrogen Metabolism, Enzymic Activity, and Mitochondria Distribution in Relation to Differentiation in the Sea Urchin Egg.” Ahnqvist and Wiksells, Uppsala. HOGEBOO~~,G. H., and SCHNEIDEN, W. C. (1950). Sonic disintegration of isolated liver mitochondria. Nature 166, 302-303. KARASAKI, S. ( 1959). Electron microscopic studies on cytoplasmic structures of ectoderm cells of the Triturus embryo during the early phase of differentiation. Embryologic 4, 247-272. KAYE, J. S. ( 1958). Changes in the fine structure of mitochondria during spermatogenesis. J. Morphol. 102, 347-400. KIELLEY, W. W. ( 1955). Mitochondrial ATPase. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, pp. 593-595. Academic Press, New York. \ eci fi c adenosine triphosphatase of KIELLEY, W. W., and KIELLEY, J. (1953). A :p liver mitochondria. J. Biol. Chem. 200, 213-222. KRUGELIS, E. (1950). Properties and changes of alkaline phosphatase activity
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during Amphibian development, Compt. rend. truv. lab. Cadsberg Sbr. chim. 27, 273-290 KUFF, E. L., HOGEBOOhf, G. H., and DALTON, A. J, (1956). Centrifugal, biochemical, and electron microscopic analysis of cytoplasmic particulates in liver homogenates. J. Biophys. Biochem. Cytot. 2, 33-54. KUTTNER, T., and COHEN, H. R. (1927). Micro-calorimetric studies: I. A molybdie acid, stnnnous chloride reagent. The microestimation of phosphate and calcium in pus, plasma, and spinal fluid. J. Biol. Chem. 75, 517531. and Physiology of LINDBERT., O., and ERNSTER, L. ( 1954). “The Chemistry Mitochondria and hlicrosomes.” Springer, Vienna. ~~ONROY, A. ( 1957). Adenosinetriphosphatase in the mitochondria of unfertilized and newly fertilized sea urchin eggs. J. Cellular Camp. Physiol. 50, 73-81. Moot, F. { 1958). Enzymes: formation and growth. In “Embryonic Nutrition” (D. Rudnick, ed.), pp. 87-102. Univ. of Chicago Press, Chicago, Illinois. Moot, F. ( 1961). The functional differentiation of the small intestine. VIII. Regional differences in the alkaline phosphatnses of the mouse from birth to one year. Develop. Biol. 3, 153-174. “Normal Table of Xenopus Zuevis NIEUWKOOP, P. D., and FABER, J. (1956). ( Daudin ) .” North-Holland Publishing Co., Amsterdam. NOVIKOFF, A. B. (1961). Mitochondria. In “The Cell” (J. Brachet and A. E. Mirsky, eds.) Vol. 2, pp. 299-421. Academic Press, New York. NOVIKOFF, A. B., PODBER, E., RYAN, J., and NOE, E. (1953). Biochemical heterogeneity of the cytoplasmic particles isolated from rat liver homogenate. J. Histochem. and Cytochem. 1, 27-46. PALADE, G. E. (195.3). An electron microscope study of the mitochondrial structure. J. Histochem. and Cytochem. 1, 188-211. PASTEELS, J. J. (1958). Comparative cytochemistry of the fertilized egg. In “Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), pp. 381-403. Johns Honkins Press, Baltimore, Marvland. REBHUN, L. I. ( 1956). Electron microscopy of basophilic structures of some invertebrate oocytes. I. Periodic lamellae and the nuclear envelope. J. Biophw and B&hem. Cutol. 2, 93-104. REVERBERI, G. ( 1957). The role of some enzymes in the development of Ascidians. In “The Beginning of Development” (A. Tyler, R. C. von Borstel, and Ch. B. Metz, eds.), DR. 319340. Publ. 48 AAAS, Washington, D. C. SCHNEIDER, W. C. ( 1953). Biochemical constitution of mammalian mitochondria. J. Histochem. and Cytochem. 1, 212233. SCHNEIDER, W. C., and HOGEBOOM, G. H. ( 1951). Cytochemical studies of mammalian tissues: The isolation of cell components by differential centrifugation: A review. Cancer Research 11, l-22. SHEN, S. C. ( 1955). Enzyme development as ontogeny of specific proteins. In “Biological Specificity and Growth” (E. G. Butler, ed.) pp. 7-S-92. Princeton Univ. Press, Princeton, New Jersey. SHEN, S. C. ( 1958). Changes in enzymatic patterns during development. In “The Chemical Basis of Development” (W. D. McElroy and B. Glass, eds,), pp. 416-432. Johns Hopkins Press, Baltimore, Maryland.
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SIEKEVITZ, I’., and ~VATSON, RI. L. (1956). Cytochemical studies of mitochondria. II. Enzymes associated with a mitochondrial membrane fraction. J. Biophys. Rio&em. Cytol. 2, 653469. SOLOMON, J. B. ( 1959). Changes in the distribution of glutamic, lactic, and malic dehydrogenases in liver cell fractions during development of the chick embryo. Develop. Biol. 1, 182-198. THOMSON, J. F., and Moss, E. M. (1956). The intracellular distribution of a bound acid phosphatase of rat liver as studied by gradient centrifugation. Arch. Biochem. Biophys. 61, 456-460. WEBEH, R. (1956). Zur Verteilung der Mitochondrien in friihen Entwicklungsstadien von Tubifex. Rev. suisse zool. 63, 278-288. \VEBER, R. ( 1958). Ueber die submikroskopische Organisation und die biochemische Kennzeichnung embryonaler Entwicklungsstadien von Tubifex. Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 150, 542-580, WEBER, R., and BOELL, E. J. (1955). Ueber die Cytochromoxydaseaktivitat der Mitochondrien von friihen Entwicklungsstadien des Krallenfrosches (Xenopvs Zaevis Daud.). Rev. suisse zool. 62, 260-268. WILSON, E. B. (1928). “The Cell in Development and Heredity,” pp. 45ff. Macmillan, New York. YCAS, IM. (1954). The respiratory and glycolytic enzymes of sea-urchin eggs. J. Exptl. Biol. 31, 208-217.
BIOLOGY,
4, 452472
( 1962)
Enzyme Patterns in isolated Mitochondria from Embryonic and Larval Tissues of Xenopus’ R. WEBER AND E. J. BOELL Department
of Zoology,
Yale Uniuersity,
Accepted
December
New
Huoen,
Connecticut
26, 1961
INTRODUCTION
During the past decade and a half, impressive studies have been made on the structural and biochemical characteristics of the mitochondria of fully differentiated cells. These cytoplasmic particles are of interest not only because of their elaborate morphological organization, but also because they serve as important centers of metabolic activity. They contain, among others, the constellation of enzymes concerned with oxidative phosphorylation (Schneider and Hogeboom, 1951; Lindberg and Ernster, 1954; Ernster and Lindberg, 1958; Novikoff, 1961) , and they are, therefore, important agents in energy transfer in cells and tissues. Furthermore, it has been demonstrated that the mitochondria derived from a given adult tissue comprise a heterogeneous population of cytoplasmic constituents (de Duve et al., 1953; Novikoff et al., 1953; Kuff et al., 1956; Thomson and Moss, 1956). And there is some evidence to suggest that mitochondria obtained from different tissues have specific biochemical characteristics (Felix et al., 1954; Green and Beinert, 1955). It has been shown, in a number of now classical studies (Wilson, 1928) and more recently through the use of the electron microscope, that mitochondria are important inclusions of eggs and embryonic cells (Weber and Boell, 1955; Weber, 1956; 1958; Eakin and Lehmann, 1957; Rebhun, 1956; Karasaki, 1959) and that specific enzymes are associated with them (Cleland, 1951; Gustafson, 1952; Yeas, 1954; Boell and Weber, 1955; Solomon, 1959). ’ This work was supported by grants from the National Science Foundation Na(G-4288 and G-8771 ) and, in part, by a g rant from the Schweizerischen tional fonds. 452
ENZYME
I’ATTERXS
IX
hlITOCHONDRIA
453
From time to time, attention has been focused on the developmental significance of mitochondria, with special emphasis on such questions as variations in their relative number, their distribution within the cell or within various presumptive regions of the embryo, developmental changes in their cytochemical and biochemical properties, changes in morphology, et cetera (Gustafson, 1959; Boell and Weber, 1955; Eakin and Lehmann, 1957; Reverberi, 1957; Kaye, 1958; Pasteels, 1958; Shen, 1958; Novikoff, 1961). Although the literature on the subject is voluminous, little or no concrete evidence exists which can help us to determine whether the specific role of mitochondria in embryonic cells is fundamentally any different from that in mature cells. As Novikoff (1961, page 367) has recently stated, “The precise role of mitochondria in embryonic development remains to be established. Apparently, mitochondria of ova and embryos possess the same fine structure as in adult cells . . . but changes may occur during development. There is little reason to doubt that most possess essentially the same oxidative, phosphorylating, and other activities as found in mitochondria of mature tissues. . . . The unanswered question is whether they serve exclusively as suppliers of energy and metabolites, or have additional, more specific directive roles in development.” In view of the results summarized above, some of the changes in enzyme activity known to occur during embryonic development (Boell, 1955; Shen, 1955; Moog, 1958; Duspiva, 1955) may reasonably be expected to reflect ontogenetic changes in the properties of mitochondria. The present study was undertaken to explore this possibility. In this approach, emphasis has been shifted from mitochondria to mitochondria as objects of as agents of embryonic differentiation embryonic differentiation. Our preoccupation with the latter aspect of the subject does not imply, however, that we discount the possible importance of mitochondria in the former. Indeed, any cellular constituent concerned directly or indirectly with protein synthesis must have significance in the differentiation process. In previous work we have been able to show a progressive increase in cytochrome oxidase activity in isolated mitochondria from embryonic and prefeeding stages of Xenopus (Boell and Weber, 1955; Weber and Boell, 1955). This was interpreted as the result of differential growth rates of the members of a heterogeneous population of mitochondria as well as intrinsic changes in individual mite-
454
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AND
BOELL
chondria. The results of recent observations of amphibian embryonic cells with the electron microscope are consonant with this view for they show that the mitochondria undergo progressive differentiation both in shape and in internal fine structure (Eakin and Lehmann, 1957; Karasaki, 1959). These findings have led us to extend our earlier experiments on the biochemical changes in mitochondria during embryonic differentiation of Xenopus. Our previous study involved only a determination of cytochrome oxidase activity in mitochondria derived from embryos of different ages. In the present work, comparative data are presented on the specific activities of four enzymes (cytochrome oxidase, ATPase, acid phosphatase’ and cathepsin) in mitochondria obtained from undifferentiated tissue primordia and from fully differentiated larval tissues. We shall try to answer the following questions: (1) Are mitochondria of undifferentiated tissue primordia characterized by specific enzyme patterns? (2) Do tissue-specific changes occur in mitochondrial enzyme patterns during differentiation? MATERIAL
Preparations
of Mitochondriul
AND
METHODS
Fractions
Mitochondria were isolated by differential centrifugation either from whole and dissected tailbud embryos or from tissues of full grown larvae of Xerwpus laevis. 3,4 An important point in our experimental design was the investigation of mitochondria from corresponding tissues at an early stage of development and at the completion of morphological and physiological differentiation. As a source of relatively undifferentiated tissue, tailbud embryos (stage 33/34, Nieuwkoop and Faber, 1956) were selected. These ‘We do not know whether or not in embryonic tissues acid phosphatase is associated with specialized cell particulates such as those derived by de Duve (1957) from adult tissue and characterized by him as “lysosomes.” If it is, we were unable to clear our mitochondrial preparations of lysosomes, for they always contained acid phosphatasc in high concentration even after repeated washing and recentrifugation, cf. page 462. ‘We are greatly indebted to Professor F. E. Lehmann, Zoological Institute of the University of Bern, Switzerland, who kindly supplied us with adult Xenopus. ‘We also wish to express our gratitude to the “Ciba” A-G Basle, Switzerland, for a sample of choriogonadotropic hormone which never failed to induce spawning.
ENZYME
PATTERNS
were placed in amphibian Ringer membranes were removed. The and the remaining body portion into a dorsal and a ventral half
IN
MITOCHONDRIX
455
solution, and the jelly and vitelline heads were cut off and discarded, of each embryo was then dissected (Fig. 1). By this procedure it was
DORSAL HALF = “MESODERM” I
I
\
“ENTRLL
HALF = “ENDODERM”
FIG. 1. Dissection of taiibud embryos. embryos were cut into dorsal and ventral “mesoderm” and “endoderm” cells.
The broken lines indicate how the halves which served as sources for
possible to collect in a relatively short time fragments of embryonic tissue which were predominantly mesodermal (dorsal halves) or endodermal (ventral halves). These fragments, or in some instances whole embryos of the same stage, were transferred to ice-cold 0.28 M sucrose + 10e3M Versene, neutralized to pH 7.2 with NaOH ( = S-Vmedium). As samples of differentiated tissue, derived from endoderm and mesoderm, respectively, liver and tail muscle of Xenopus larvae at the beginning of metamorphosis (stage 56/57, Nieuwkoop and Faber, 1956) were taken and, after transfer to chilled S-V-medium, were freed from contaminating tissues. Homogenates were prepared at low temperature by grinding tissue samples in a conical all-glass microhomogenizer with a small amount of S-V-medium. The tissue brei was then further diluted with S-Vmedium, giving concentrations of 10 dorsal or ventral fragments per 100 ~1 homogenate and 10 mg liver or tail tissue per 100 ~1 homogenate. Since our main interest lay in the biochemical properties of mitochondria, we did not attempt any complicated fractionation pro-
456
WEBEH
AND BOELL
cedure, but followed our earlier method which with some minor modifications was found satisfactory. The homogenates were fractionated by means of a microcentrifuge with a head accommodating 12 horizontally rotating tubes, each tube measuring 28 mm in length and having a capacity of 0.5 ml. In order to reduce heating effects, due to high speed centrifugation, the centrifuge was operated in a refrigerator at a temperature of O-5” C. This measure was effective, except for runs at top speed during which the tubes were slightly heated. In these cases, however, exposure to slightly elevated temperature lasted a few minutes only and hence could not be too deleterious to the mitochondrial preparations. Table 1 shows a survey of the isolation procedure by which relaTABLE I FRACTIONATION PROCEDURE
300 ~1 in 0%
Homogenate :I[ sucrose + 1O-3 !lI Versene 10 min . . 700 q 1
1
It, : Yolk, pigment granules, tissue debris + 300 ~1 S-V-medium I I 10 min . TOO q 1 SW RIW + 300 ~1 S-V-medium I 10 min . .
iO0 q
I SW RIW ’ + 150 ~1 S-V-medium = Fraction 1: Yolk, pigment granules, tissue debris * Supernatants
SW + SW’ I I I 1* I* R2h R,, ---_--
Sl I 1 15 min
. . 17,500 g
A Ran 2
k? s I + 300 ~1 S-V-medium / I I 1 15 min . . . 17,500 g /
1* /\ l&w Rz L_-+ ca. 150 ~1 S-V-medium
S, Final supernatan,i including microsomes
= Fraction 2: Mitochondria, some pigment granules
from these washings were discarded.
‘The microcentrifuge was designed Hohnevej, Brendby Strand, Denmark.
and
constructed
by Mr.
Ole
Dich,
18
ENZYME
PATTERh-S
IN
MITOCHONDRIA
457
tively pure mitochondrial preparations could be obtained in a rather short time. The volumes of the homogenate were measured by means of constriction pipettes and those of the fractions were determined by weighing. The data of the distribution of total nitrogen showed that in spite of the small amounts of starting material excellent recoveries and well reproducible values were obtained, thus indicating only slight losses of material during differential centrifugation. Enzyme
Assays
The activities of cytochrome oxidase, ATPase, acid phosphatase, and cathepsin were determined by means of micromethods which will be described in detail below. For each enzyme studied, a series of experiments was performed on the assay technique alone in order to make sure that determinations of enzyme activity were done under optimal conditions. In some experiments, the enzyme activities of all fractions including the washings were determined. In most cases, however, the activities were determined only on homogenates and isolated mitochondria. It should be noted that the relative activities of enzymes in mitochondrial preparations were found to vary somewhat from experiment to experiment, probably because of biological variability in the embryos obtained from different females. Cytochrome oxidase activity was determined at 25” C by means of the Cartesian diver technique, using siliconed divers of approximately 10 ~1 total volume. The diver filling consisted of: 0.5 ~1 l/10 N NaOH ( bottom drop), 0.4 ~1 substrate (side drop, containing 0.1 M p-phenylenediamine and 2.5 x lo-” M cytochrome c in 0.1 M phosphate buffer of pH 7.4), 0.6 ,~l homogenate or mitochondrial suspension (neck seal), 0.7 ,J paraffin oil, and mouth seals measuring about 3 mm in length. After a period of temperature equilibration, usually 10-20 minutes, enzyme and substrate were mixed by raising the pressure in the diver system, and the reaction was then followed for a l-hour period. The readings were taken at intervals of 5-10 minutes, and in most cases the oxygen uptake was at a linear rate, at least during the first 30 minutes. The concentration of the enzyme samples was adjusted so that the pressure change in the divers ranged between 60 and 120 mm per hour. Final readings were corrected for substrate autoxidation, but, as was true in our previous work, endogenous respiration of homogenates and of mitochondrial suspensions was negligible.
458
WEBER
AND
BOELL
Mitochondrial ATPase activity was assayed, following in part the methods of Kielley (1953, 1955) and Monroy (1957). To 5 ,J of substrate ( ATP-disodium-salt” 1.5 X lo-” M + MgCl, 1.5 X 10e3M barbital buffer 0.1 M pH 7.6), 5 ~1 of the enzyme sample was added and incubated for 30 minutes at 25” C. The reaction was stopped by adding 10 ~1 of chilled 5% trichloroacetic acid + 0.5% uranium sulfate (Krugelis, 1950). The tubes were immediately put into a refrigerator at 0” C, in order to avoid acid hydrolysis of ATP. As controls, substrate samples were precipitated before addition of the enzyme aliquots. Thus a correction was made only for free PO,--- present in the enzyme samples, since it was found that, by working at low temperature, spontaneous breakdown of ATP in presence of TCA was negligible. The liberated PO,--- was estimated according to the sensitive calorimetric method of Kuttner and Cohen (1927). The chilled tubes were centrifuged for 5 minutes at 17,500 g and then 10 ~1 of the supernatant was removed. To this aliquot, 10 ~1 each of 4 N HZSOI, 3% ammonium molybdate, and 0.8% &Cl, were added in the order stated, with thorough mixing of the content of the tubes after each addition. The blue color was allowed to develop for 30 minutes and then readings were taken on samples of 20 ~1 at 640-700 rnp (Klett Filter No. 66) by using a microcolorimeter similar to the design of Krugelis (1950). In a series of test readings our apparatus showed an excellent agreement with values obtained from the Beckman DU spectrophotometer at 670 mp. It should be mentioned that the PO,-reaction is extremely sensitive to impurities, which lead to considerable differences in color intensity between duplicate samples. This difficulty can be overcome, however, by a careful cleaning procedure of the tubes, in which they are first immersed in hot cleaning solution (sulfuric acid-sodium dichromate) then rinsed at least five times with glass-distilled water. Bv this means it is possible to reduce variations among triplicates to less than 5%. ATPase activity was expressed as micromoles P X 10m3liberated per microgram TN in 30 minutes (specific activity) and as micromoles P X lo-” liberated per 30 minutes (total activity). Acid phosphatase activity. To 5 ~1 of substrate (3.1 X 1O-2M ‘Obtained
frown the Sigma Co., St. Louis, Missouri.
ENZYME
PATTERNS
IN
MITOCHONDRIA
459
sodium-/&glycerophosphate’ + 0.06 211acetate buffer pH = LO), 5 ~1 of the enzyme sample was added, and, after mixing, the tubes were incubated at 39” C for 4 hours. The reaction was stopped by addition of 10 ~1 of chilled 5% trichloroacetic acid (TCA) + 0.5% uranium sulfate and then the tubes were put into a refrigerator at 0” C. AS controls, substrate aliquots were mixed with chilled TCA and to these tubes the enzyme samples were added. Since it was found that in the presence of TCA neither substrate nor homogenate samples released appreciable amounts of P04m--, only a correction for endogenous free PO,--- was made. The calorimetric determination PO,--- was the same as that described for ATPase assays. The activity of acid phosphatase was expressed as micromoles P X lo-” released per microgram TN per 1 hour (specific activity) and as micromoles P x 10m3released per 1 hour (total activity ). C&epic activity was determined according to the calorimetric method of Duspiva (1939) with a mixture of 2% casein “Hammarsten” as substrate in 48% urea and citrate-phosphate-ammonia buffer pH = 4.80. The reaction mixture which consisted of 10 ~1 of substrate and of 5 ~1 enzyme aliquots was incubated for 3 hours at 37” C. The reaction was stopped by adding 100 ~1 of chilled 5% TCA, and then the tubes were left standing for at least 4 hours in the refrigerator. The controls were set up by adding the enzyme to the tubes which contained substrate and TCA samples. For the calorimetric determinations the precipitated reaction mixtures were centrifuged for 5 minutes at 17,500 g; 50 PI of the supernatant was thoroughly mixed with 50 ~1 of 13.5% Na,CO, (anhydrous), and after addition of 50 ,.J Folin-Ciocalteu-reagent (diluted 1:5), the blue color was allowed to develop for 30 minutes. Again, the calorimetric readings were taken on 20-J aliquots by means of the microcolorimeter using a Klett-Filter No. 69 with a spectral range of 660-740 rn&, The enzyme activities were compared to a standard activity curve, obtained from a dilution series of a liver homogenate in S-V-medium. This curve showed a linear relationship between activity and homogenate concentration up to 0.5 mg liver tissue per 100 J homogenate. By means of a standard curve the E values were converted into micrograms casein per 10 ~1 and the activity units for cathepsin were ’ Obtained from the Sigma Co., St. Louis, Missouri.
460
WEBER
AND
BOELL
then expressed as micrograms casein split per microgram TN per hour (specific activity) and as micrograms casein split per hour (total activity ) . All determinations were made in duplicate; the variation between them was found to be less than 5%. Total nitrogen ( TN) was determined by means of an ultramicroKjeldahl method (Boell and Shen, 1954) on duplicate samples that contained 1-15 pg TN. Electron
Microscopy
In order to obtain complementary information on the morphology of the mitochondria that were studied biochemically, samples of all the tissues which served as sources for particulate fractions were observed by means of a Siemens “Elmiskop l”* working at 60 kv and 5 pamp. RESULTS
The Yield of Enzyme Activity
from
Mitochondrial
Preparations
By comparing specific enzyme activities in mitochondrial preparations and in corresponding homogenates, we obtained activity ratios indicating the extent to which the enzymes are concentrated in the mitochondria. The data, summarized in Table 2, show that the acTABLE
2
CONCENTRATION OF ENZYMES IN MITOCHONDRIA (WHOLE HOMOGENATE = l)a ATPave
Whole embryos (gastrula-tailbud) “Mesoderm” halves (tailbud) “Endoderm” halves (tailbud) Liver (larval stage 55) Tail muscle (larval stage 55)
8.2 7.0 12.0 2.9 8.2
Mammalian liver tissue (from Schneider, 1953)
3.1
a (l), (2), (3), (4) = respectively,
(4) (3) (3) (3)
7.1 5.2 6.2 3.1
(4) (3) (3) (1)
(1)
3.9
(1)
1 experiment
2.4
Acid phosphatase
4.2 3.5 3.8 1.5 6.7 1.3
(3) (3) (3) (1) (1)
Cathepsin
4.5 3.8 6.0 2.0 6.2
(4) (3) (3) (2) (1)
2.6
or means of 2, 3, or 4 experiments.
*We are greatly indebted to the Electron Microscopy Division of the Institute of Inorganic Chemistry at the University of Bern for assistance in operating the electron microscope.
ENZYME
PA’ITERNS
IN
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461
tivities of all of the enzymes studied are considerably greater in mitochondria than in homogenates. The table shows the further extremely interesting point that the degree to which the enzymes are concentrated is different for each of the four enzymes tested and also differs for each of the tissues from which the mitochondria were derived. It seems clear, therefore, that mitochondria from one embryonic tissue cannot be regarded as equivalent to those from another. The data suggest that the pattern of enzyme concentration, as well as the relative activities of the four enzymes studied (Table 3), comprise a set of biochemical characteristics specific for each kind appealing of embryonic or adult tissue. Such an idea is particularly when the remarkable similarity in the enzyme activities of Xen~pus liver and mammalian liver are noted (Table 2). Although the data are not numerous, the similarity in the activity ratios seems to be altogether too close to be purely fortuitous. It was not our intention to make an elaborate analysis of enzyme distribution in the various fractions obtained through differential centrifugation of tissue homogenates. A few measurements of this kind were made, however, so that the total activity of homogenates which could be accounted for by the activity of the mitochondrial fractions could be determined. In general, the relative recovery of enzyme activity in mitochondria in our experiments agreed well with data reported in the literature ( Schneider, 1953)) although absolute recovery was occasionally somewhat lower. This is undoubtedly due to minute losses of mitochondria during the isolation procedure. It may be recalled that, in comparison with the usual techniques for isolating mitochondria, we were working with extremely small volumes of homogenate and particulate suspensions ( microliters instead of milliliters ) . Small losses, insignificant in the usual macro techniques, would thus have a considerable effect on recovery of enzyme activity. In the case of cytochrome oxidase, no appreciable amount of enzyme was ever found in nonmitochondrial fractions. Yolk and tissue debris (R,), the final supernatant (S,), or the washings never showed more than 6% of the activity of the original homogenate. On the other hand, in the case of ATPase, acid phosphatase, and cathepsin, an appreciable percentage of the total activity of the tissue was recovered in the final supernatant. The recoveries in the supernatant on the average amounted to 20% for ATPase, whereas for acid phos-
462
WEBEH
.&SD
UOELL
phatase and cathepsin occasionally as much as 50% could be recovered in the supernatant. This finding is of interest, for on a quite different material, viz., mammalian liver, sedimentation behavior for particles that carry cytochrome oxidase was discovered to be different from that of particles to which acid phosphatase and cathepsin are bound (de Duve et al., 1953). Whether this also applies to particles derived from embryonic and differentiated cells of Xenopus is not known at present. Recent findings on the development of glutamic dehydrogenase activity in differentiating liver tissue of chick embryos, after the 12th day of incubation, indicate inversely changing percentage activity in mitochondria and in the supernatant (Solomon, 1959). To what extent ATPase, acid phosphatase, and cathepsin, found in differentiating tissues of Xen,opus, reflect a similar phenomenon cannot be decided from our data. Cytochrome Oxidase Activity and Differentiated Cells
in Mitochondria
from Tissue Primordiu
The results on cytochrome oxidase will be considered first, since they bear directly upon our main problem, namely the biochemical heterogeneity of mitochondrial populations. In Fig. 2 a comparison is made of the cytochrome oxidase activity per unit of TN for mitochondrial fractions obtained from whole embryos, dorsal halves ( = “mesoderm”) and ventral halves ( = “endoderm”) of the tailbud stage. It may be noted that “mesoderm” mitochondria contain 3.25 times more activity per unit nitrogen than do mitochondria isolated from “endoderm”; the mitochondria from whole embryos show an intermediate level of activity. Now if “mesoderm” and “endoderm” are compared with their derivatives, namely larval tail muscle and liver, one is struck by the fact, that the specific activities of these mitochondria are almost identical with those of the corresponding tissue primordia. Thus the activity ratio for tail muscle:liver amounts to 3.1:I. One may conclude that during development mitochondrial populations from relatively undifferentiated cells ( tissue primordia ) may already show differences in their enzyme content. This result has an important implication which bears upon the fractionation procedure. In view of the different properties of the it is unlikely that equal homogenates which were fractionated, amounts of contaminating “inert proteins” were present in the mito-
ENZYME
PATTE:RIVS
SPECIFIC ACTIVITY
IN
463
MITOCHONDRIA
3.25
2500 Moo 1500 1000 500 0
I
1
EMBRYO
WHOLE EMBRYO \ ‘\
EMBRYO “ENDODERM” FIG. 2. Cytochrome and differentiated larval pg TN.
TAIL MUSCLE
“MESODERM”
LIVER
oxidase in mitochondria of undifferentiated embryonic tissue. The units of specific activity are 10e3 ~1 Oz/hr/
chondria from embryonic tissues (“mesoderm” or “endoderm”) and from differentiated tissues (tail muscle or liver) and that such contamination was responsible for the figures reported in Figs. 2 and 3. Instead, it seems reasonable to regard the observed differences in specific activity as true biochemical characteristics of these mitochondrial preparations. Patterns of Enzyme Activity
in Mitochondriu
of Various
Tissues
Table 3 presents average figures for the specific activity levels cytochrome oxidase, ATPase, acid phosphatase, and cathepsin mitochondria from the various embryonic and larval tissues used this study, i.e., from whole embryos, endoderm, and mesoderm stages 33/34 and from liver and muscle dissected from larvae stages 56/57,
of in in at at
464
WEBER
AA-D
BOELL
TABLE 3 SPECIFIC ACTIVITY~ OF ENZYMES IN MITOCHONDRIA FROM EMBRYONIC AND LARVAL TISKES OF SENOPIJ~ Cytochrome oxidase
Acid
ATPase
phosphataue
CathepGn
Q
R
Q
R
Q
R
Q
R
Tailbud embryo Dorsal half “mesoderm” Ventral half “endoderm”
1038
1.0
4.55
1.0
0.70
1.0
7.18
1.0
2493
2.4
8.86
1.9
2.38
3.4
10.56
1.5
768
0.7
5.52
1.2
0.76
1.1
5.22
0.7
Tail muscle
2030
2.0
18.5
4.1
0.72
1.0
7.07
1.0
649
0.6
32.3
7.1
Liver
10.5
15.0
35.8
5.0
a Q = specific activity; R = ratio of activity compared with tailbud embryo. For description of activity units, see Methods.
The table shows that the specific activities of all the enzymes tested are different from tissue to tissue. It is also apparent that developmental changes in enzyme activity within the mitochondria of a given tissue may involve ( 1) no significant alteration in level, (2) an increase, or (3) a decrease in activity. Furthermore, the figures show that during development the variations in activity of a particular enzyme within the mitochondria of a given tissue are completely independent of those of any of the other enzymes, both in terms of direction and magnitude of change. With cytochrome oxidase, for example, activity declines approximately 15% during development in both liver and muscle. ATPase activity increases in both tissues, but in muscle it does so by only a little more than twofold, while in the differentiation of liver, activity rises by almost sixfold. By contrast, acid phosphatase declines during differentiation of muscle, but during the same developmental period it increases very considerably (fourteenfold) in the liver. The developmental change in catheptic activity is similar in direction to that of acid phosphatase, decreasing in muscle and increasing in the liver, but the extent of change of cathepsin activity is quantitatively quite different from that of acid phosphatase. From a consideration of such data, it appears that the development of a specific pattern of enzyme activity is typical of the mitochondria from each of the tissues studied. The distinctive character of the
ENZYME
PATTERNS
IN MITOCHONDRM
465
changes in pattern of enzyme activity, occurring during morphological and functional differentiation of muscle and liver, are illustrated in Fig. 3. 20-
/ /V,
IO-= 51 -
-
0' /'LIVER /I
. .
-0
ENDODERM
TAIL MUSCLE MESODERM
FIG. 3. Enzyme patterns in mitochondria of undifferentiated embryonic and differentiated larval tissues. The activity levels are plotted in a logarithmic scale which indicates units of specific activity. The figures for ATPase, acid phosphatase, and cathepsin are multiplied by 100 to bring them into convenient scale.
DISCUSSION
In tracing changes in the enzyme activity of mitochondria, during the development of a tissue from its primary germ layer antecedent to the fully differentiated state, it would be desirable to make measurements not only on stages at the beginning and at the end of the developmental period under consideration, but also on a number of intermediate ones as well. Unfortunately, this could not be done in the present study owing largely to still unresolved difficulties in isolating and fractionating the extremely small bits of tissue which embryonic tissues and organs represent, especially in early stages
466
WEBEH
AND
BOELL
Hence, the present study must be regarded as somewhat exploratory rather than comprehensive. But in spite of this, several features in the results obtained stand out clearly and are worthy of further comment. First, it should be emphasized that mitochondria from different tissues, even from the earliest embryos tested, have distinctive characteristics which are revealed in significant quantitative differences in specific activity of particular enzymes. This is shown graphically in Fig. 2 for cytochrome oxidase and in Table 3 for the other enzymes tested. A further point of interest is the fact that the “developmental behavior” of each of the enzymes appears to be unique for a given tissue. This is best seen in comparisons of the activity of a particular enzyme in mitochondrial suspensions from the early whole embryo, from a tissue primordium, and from the differentiated derivative of the primordium (Table 3). For example, the ratio of cytochrome oxidase activity in whole embryo, mesoderm, and tail muscle 1:2.4:2. By contrast, for ATPase activity from the same three sources, the ratio is 1:1.9:4.1. The ratio of cytochrome oxidase activity in early whole embryo, endoderm, and liver is 1:0.7:0.6; for ATPase, it is 1:1.2:7.1; and for acid phosphatase it is 1:1.1:15. These calculations provide the most powerful argument we know against the suggestion that quantitative differences in specific activity, such as those noted, are due to variations in the degree of contamination of the mitochondrial preparation with nonenzymatic constituents. The observed difference in the specific activities of mitochondria from various tissues helps to answer a question which we raised in our earlier study of cytochrome oxidase activity in mitochondria obtained from whole Xenopus embryos (Boell and Weber, 1955). In commenting on the rise in cytochrome oxidase activity per unit of mitochondrial nitrogen which occurs during development, we stated: “The change in the Q value of mitochondria might represent something as simple as the gradual disappearance during development of a contaminant of the mitochondrial fraction. It might also reflect a shift in the ‘population statistics’ of the mitochondrial fraction representing changes in the relative numbers of specific kinds of mitochondria brought about by differential growth of the various tissues of the embryo. On the other hand, it could represent an intrinsic
ESZYME
PATTERNS
IN
MITOCHONDRIA
467
change in the individual mitochondria characterized by the transformation of catalytically inert protein into cytochrome oxidase.” From the discussion above, it seems to us very unlikely that the presence and gradual disappearance of a contaminant in the mitochondrial preparations could in any way account for the changes in specific activity of enzymes reported in this paper. Furthermore, although the possibility of intrinsic changes occurring in mitochondria during development is by no means ruled out, nor is it even lessened, it is now clear that the development of a heterogeneous population of mitochondria must account, to some extent-perhaps to a considerable extent-for the facts observed in the work referred to above. Mitochondria from mesoderm and muscle tissue, as can be seen from Fig. 2, have a threefold greater specific activity of cytochrome oxidase than mitochondria from endodermal tissue. Hence, the progressive increase in the proportion of muscle in the embryo as development proceeds would be expected to lead to a rise in specific activity of mitochondria from homogenates of the whole embryo. Relatively more of the particulates in such a suspension would represent mitochondria contributed by muscle tissue. Any attempt to evaluate the significance of changing patterns in enzyme activity during development leads one to ask whether the observed results are due simply to increases or decreases in the number of units of enzyme in a particular tissue or whether enzyme molecules themselves are undergoing change as development proceeds.” Usually the dynamic properties of enzymes are defined by the Michaelis-Menten constant, K,,,, for a given substrate. We applied this method (\R’eber, unpublished) in an attempt to assess the properties of acid phosphatase which, as will be seen in Table 3, undergoes the most impressive developmental change in activity of the four enzymes tested. Although this work is still in somewhat preliminary form, the main result is summarized in Table 4 and shows that K, is almost constant for mitochondria from embryonic and differentiated tissues (compare Moog, 1961). Many factors, especially the purity of the enzyme preparation, can affect the Michaelis-Menten constant, but, if the results summarized in Table 4 are taken at face value, there seems to be no change in ‘This view has been advanced sion of Moog’s paper ( 1958).
by de Villafranca,
among others,
in a discus-
468
WERER AND
BOPLI.
TABLE 4 MICHAELIS-MENTEX
COSSTAKT AT DIFFERENT
Source of enzyme
(Km)
extract
Whole tailbud embryo Larval liver Larval tail muscle
OF ACID
STAGES
PHOSPHATASE
Km x 10-z * &I<.,,
3.10
3.39 3.80
FROM TISSUES
OF DEVELOP.MENT ,V”
0.81 0.38 0.33
3
14 G
a S.E. = standard error. b N = number of determinations.
affinity of acid phosphatase for its substrate. It would seem, therefore, that the developmental change in acid phosphatase activity is due to an increase in the number of enzyme molecules rather than to changes in the enzyme, as such. In view of the striking differences which have been demonstrated in the enzyme activity of mitochondria from mesoderm and muscle, on the one hand, and endoderm and liver, on the other, the question arises whether any correlation exists between the biochemical findings and the structural organization of these cytoplasmic constituents. It was shown for rat liver mitochondria that cytochrome oxidase, and certain other enzymes, are firmly bound to mitochondrial structure (Hogeboom and Schneider, 1950). More recently, Siekevitz and Watson (1956) have reported that when mitochondria are disrupted oxidase and succinoxidase remain by deoxycholate, cytochrome firmly attached to a fraction identified as consisting of mitochondrial membranes. In view of these observations, it is of interest to note that preliminary results of an electron microscopic study of Xenop~ larval tissues reveal a difference in the fine structure of mitochondria from tail muscle and from liver. In mitochondria from tail muscle, there is an elaborate system of internal cristae with relatively little space for matrix. In liver mitochondria, on the other hand, the cristae are much less numerous and seem to be confined to the periphery, thus leaving a large volume of mitochondrial ground substance or matrix. Similar differences in fine structure have been shown to exist in mitochondria of striated muscle and the parenchymatous cells of the liver in the adult rat (Palade, 1953). It appears, therefore, that the quantitative differences in specific activity of cytochrome oxidase are correlated with morphological differences in the mitochondria. In
ENZYhlE
PATTERNS
IN
h’flTOCHONDHIA
469
view of this, it would be particularly interesting to examine mitochondrial fine structure in a tissue in which cytochrome oxidase and succinoxidase activity increase considerably during development. If enzyme content is positively correlated with the quantity or degree of elaboration of cristae in mitochondria, ontogenetic changes in mitochondrial fine structure might reasonably be expected. It is difficult to assess the developmental significance of the patterns of enzyme activity in mitochondria from embryonic tissues. We do not believe that they are merely fortuitous. At the very least, they constitute biochemical indexes of the functional differentiation of embryonic tissues. They must, furthermore, represent adaptations to their specific physiological requirements. Perhaps they are intimately concerned with developmental processes, but exactly how they may be involved remains a matter for speculation. In any case, further investigation of the specific enzymatic capital of developing tissues and of the fundamental factors which control the synthesis of enzymes and which regulate their structural organization within the cell will continue to provide a challenging and fruitful field of inquiry.
The specific activities of cytochrome oxidase, ATPase, acid phosphatase, and cathepsin in homogenates of embryonic and larval tissues of Xenopus and in mitochondria derived from them have been determined. In all cases, determinations of enzyme activity were made on homogenates and mitochondria from early whole embryos, from tissue primordia, and from the fully differentiated end products of the primordia. The mitochondria from each embryonic or larval tissue studied have distinctive characteristics which appear as significant quantitative differences in specific activity of each of the four enzymes tested. During development, the changes in activity of each enzyme in the mitochondria from a given tissue follow an individual pattern which is independent of the pattern of other enzymes in the same mitochondria. In the case of cytochrome oxidase, a correlation has been found between the fine structure of the mitochondria and the level of enzymatic activity. Liver mitochondria have few cristae and relatively low specific activity of cytochrome oxidase; muscle mitochondria have numerous cristae and a high specific activity of cytochrome oxidase.
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