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The comparative biochemistry of developing Ascaris eggs
ARCHIVES
OF
BIOCHEMISTRY
AND
The Comparative
BIOPHYSICS
106, 223-228 (1964)
Biochemistry
V. Changes
of Developing
in Catalase
Activity
L. C. COSTELLO From the Department
AND
during
January
Eggs
Embryonation’
w. SMITH2
of Physiology, School of Pharmacy, Baltimore, Maryland Received
Ascaris
University
of Maryland,
28, 1964
Catalase activity was identified in homogenates of eggs of Ascaris lumbricoides var. suum. The specific activity of catalase (pmoles Hz02 decomposed per minute per milligram protein) was 30, 16, and 54 in the l-cell, 1st stage larval, and 2nd stage larval eggs, respectively. The initial catalase activity observed in fertilized unembryonated eggs (l-cell) remained constant through cleavage and gastrulation until development of the 1st stage larvae, at which time activity markedly decreased (50%). When 1st stage larvae molted to 2nd stage larvae, an immediate and marked increase (over 300yc) in activity resulted. The relationship of catalase, cytochrome oxidase, and respiration in embryonating eggs is discussed. Catalase activity through all stages of embryonation was in great excess of theoretically formed H202, apparently serving as a protective mechanism against peroxide toxicity.
In previous studies (1, Z), changes in the terminal electron transport system during embryonation of Ascaris eggs were demonstrable. In initial unembryonated eggs (fertilized uterine eggs), the authors proposed that oxygen consumption was mediated in the absence of sufficient cytochrome c oxidase activity possibly via flavin oxidase. This proposed pathway would result in the formation of H20z and would require the presence of a mechanism for the removal of peroxide. The present report provides evidence for the presence of eatalase activity in initial unembryonated eggs and a comparison of the activity during embryonation. MATERIALS
AND
METHODS
Fertilized unembryonated eggs were harvested and cultured as previously described (1, 3). Washed eggs were suspended in potassium phos1 This investigation was supported by NSF research grant G-23313 and by NASA grant NsG436. 2 Predoctoral Trainee, P.H.S. grant DT-46, in conjunction with the Department of Histology and Embryology, School of Dentistry. 223
phate buffer (0.05 M, pH 7.0) and homogenized with a French Pressure Cell under a force of 16,000 pounds. The homogenate was centrifuged at 4009 for 2 minutes and the resulting supernatant fluid (experimental homogenate) passed through glass wool. Such preparations contained 10-20 mg protein per milliliter. Immediately prior to the assay, the homogenate was further diluted (1:lO) with phosphate buffer (0.01 M, pH 7.0). All steps were performed in the cold. Protein was determined by the method of Lowry et al. (4). The manometric determination of catalase activity was performed by conventional Warburg techniques (5). Single-sidearm flasks (approx. 17 ml capacity) were utilized, and the gas phase was air. The complete system generally contained 1.0 ml homogenate, 2.1 ml phosphate buffer (0.01 M, pH 7.0) and 0.1 ml H202 (0.2 M). The temperature was 18” or 28°C. and the shaking rate was 100 strokes per minute. After 8 minutes equilibration, the H202 was tipped into the main compartment, and the oxygen that evolved was measured. The spectrophotometric assay of catalase activity was performed by “tracking” the decomposition of Hz02 at 230 mp (6,7). Assays were performed in l-cm cuvettes with a Hitashi/PerkinElmer spectrophotometer. Generally, the reaction system contained 0.3 ml homogenate, 20 pmoles
224
COSTELLO
HzOz, and phosphate buffer (0.01 M, pH 7.4) to adjust the volume to 3.0 ml. The peroxide was added with a blow-out pipette immediately prior to tracking. The blank contained homogenate and buffer. The iodine-titration assay of catalase activity was performed as described by Herbert (8). The reactions were conducted in test tubes (6 X 1 inch). The complete system contained 5.0 ml HIOz (0.02 M prepared in 0.01 M phosphate buffer, pH 7.0) and 1.0 ml homogenate. The reaction was terminated at various intervals by the addition of 2.0 ml 1 N HzS04. The remaining Hz02 was determined by titrating the entire contents of each tube in situ with thiosulfate as described (8). RESULTS
The preliminary identification of catalase activity in homogenates of eggs was established by manometric techniques. Figure 1 demonstrates the evolution of 02 from HzOz in the presence of unembryonated egg homogenate. With heated homogenate (5 minutes in a boiling water bath) no gas production was observed. The addition of cyanide (5.0 pmoles) completely inhibited the activity. In the absence of HzOz, no gas production by homogenate was detected. The results demonstrated the catalatic effect of homogenates. Furthermore, the reaction rates at 18” and 28” demonstrated 2107
0
o TEMC! 28OC
I2
3 4 5 6 7 8 910 TIME (Minutes)
FIG. 1. The evolution of oxygen from hydrogen peroxide by unembryonated egg homogenate. The system contained 20 pmoles Hz02.
AND SMITH
only a slight increase in activity with temperature which is similar to the properties of catalase (7). Of particular significance was the stoichiometry of the reaction. The 9.5 pmoles O2 evolved accounted for essentially all of the Hz02 (20 pmoles) added to the system. The decomposition of HzOz by unembryonated egg homogenates was apparently the result of catalase activity with little or no peroxidatic activity involved. Identical conditions and properties were observed when homogenates of embryonated eggs were employed. However, this assay procedure was inadequate for the determination of specific activities of catalase. Under identical conditions manometrically determined activities were approximately 16 % of titrimetrically established values. Such limitations of gasometric procedures for catalase have been described and reviewed by others (6). The spectrophotometric determination of catalase activity in homogenates of unembryonated eggs is demonstrated in Fig. 2. Because of the high activity the disappearance of HzOz could be assayed directly in these preparations. The rate of decomposition of Hz02 was stimulated by increasing the concentration of homogenate or substrate. The reaction was inhibited by adding cyanide or aside to the system or by heating the homogenate (5 minutes in a boiling water bath). While the spectrophotometric assay successfully provided further evidence of catalase activity, the procedure was limited by the turbidity of the homogenate. For example, more concentrated homogenates were too turbid for assay, and lower concentrations did not provide adequate activity for measurement. Accurate and reproducible specific activities under these conditions could not be determined, particularly with different homogenate preparations. The results of a typical titrimetric assay of catalase activity in egg homogenates are presented in Fig. 3. Catalase activity was highest during the initial l&second interval. Furthermore, some assays were conducted with lo-second intervals which also resulted in decreasing activity with time. Because of the difficulty in manipulation for lo-
IN ASCARIS
CATALASE
.I2 .I I
225
EGGS
o ~O/JMOLES l IO~MOLES X 30pMOLES
o 0.4 I’ll1 HOMOGENATE l 0.3 ITli. HOMOGENATE X 0.2 t-tll. HOMOGENATE
/
.I0
/x
I
0
I
I
I
I
I
I
I
I
I
I
I
20 40 60 80 100 120 140
20 40 60 80 100 I20
TIME (Seconds) FIG. 2. The spectrophotometric determination of the decomposition of hydrogen peroxide by unembryonated egg homogenate. On the left the concentration of homogenate was varied, and the system contained 20 pmoles of hydrogen peroxide. On the right the concentration of hydrogen peroxide was varied, and 0.3 ml homogenate was used. The homogenate preparations contained 1.82 mg. protein per milliliter homogenate.
401
35 30 25 20i
X ONE-CELL EGGS 0 IST STAGE LARVAL EGGS /" X
/
X
/
X
second readings, subsequent assays were performed by using 15second intervals. The adoption of this titrimetric procedure for determination of catalase activity of homogenate provided excellent reproducibility. In all experiments, the systems were prepared in triplicate and the repeated titration values of thiosulfate agreed within 0.1 ml. Furthermore, the homogenate preparations did not interfere with the iodine titration nor result in any “blank” H202 values. The catalase activity as determined titrimetrically was inhibited by heating the f
0
I5 30 45 60 75 TIME (Seconds)
FIG. 3. The titrimetric assay of catalase activity in homogenates of l-cell eggs and 1st stage larval eggs. The system contained 100 rmoles hydrogen peroxide and 1.0 ml homogenate in a final volume of 6.0 ml. The homogenate of l-cell eggs contained 1.44 mg protein per milliliter homogenate and the 1st stage larval preparation contained 0.94 mg protein per milliliter homogenate.
226
COSTELLO
AND
homogenate or by adding cyanide or azide. Having established these relationships, the specific activity (pmoles HzOz decomposed per minute per milligram protein) of homogenate preparations was determined from the initial 15second interval by using 1.0 ml diluted homogenate (containing 1.0-2.0 mg protein) and 100 ctmoles of HzOz. Under these conditions activity was essentially proportional (within 10 %) to homogenate concentration. The catalase activities of some different homogenate preparations are presented in Table I. The results demonstrated significant differences in fertilized unembryonated eggs (l-cell stage), 1st stage larval, and 2nd TABLE THE
OF
Specific activity (,~nwles H20~ decomposed/ minute/milligram protein)
Observation No.~
Mean
stage larval eggs. The 1st stage larval eggs exhibited only 50% of the activity observed initially, whereas activity was markedly increased in eggs containing 2nd stage larvae. The means and standard deviations demonstrated the excellent reproducibility in different preparations. Having demonstrated these significant changes in catalase activity of l-cell, 1st stage larval, and 2nd stage larval eggs, investigations were conducted to determine the activity during embryonation (Table II). The initial catalase activity remained relatively constant through cleavage to the tadpole stage. However, activity rapidly declined (50 %) at the time of development of the 1st stage larvae and remained constant throughout this stage. Immediately following molting, the activity increased (200 %) in the 2nd stage larvae.
I
CATALASE ACTIVITY OF HOMOGENATES DIFFERENT STAGES OF ASCARIS EGGS
1 2 3 4 5 6 =k SD
DISCUSSION
l-Cd state stage
1st Stage Ianal eggs
2nd Stage larval eggs
25.6 32.0 25.6 32.0 30.6 33.2 29.8 f3.4
14.0 16.0 16.4 18.0 15.0 15.8 f1.6
52.0 54.0 57.2 60.0 46.8 54.0 f5.2
= Each observation represents tivity of a different preparation.
the catalase
The properties of catalase and the difficulties and conditions for its assay in various types of preparations have been recently and extensively reviewed (6). Due to the use of crude preparations in the present report, catalase activity was determined by three different assay procedures. The successful incorporation of the manometric, spectrophotometric, and titrimetric assays demonstrated the presence of catalase in Ascaris egg preparations. Furthermore, the stoichiometric results obtained by the manometric assays indicated the absence
ac-
TABLE CHANGES
IN
CATALASE ACTIVITY Specific
Culture
SMITH
l-Cell
2-4 Cell
activity
(qnoles
Gastrula-
$$f;
II
DURING EMBRYONATION
tadpole
1
32.0
26.0
-
-
2 3
30.6 32.0
28.4
24.0
32.0 26.2
4 5
-
-
28.8
27.2
Hz02
OF ASCARIS EGGS
decomposed/minute/milligram
protein)
2nd Stage larval0 eggs 1st stage
larvala
eggs Early
14.0 15.0,
17.6
b
Late
52.0
70.0,
-
46.8 -
16.4, 16.4, 18.0, 32.0” 13.2
57.2 -
60.0,
64.2 60.0 -
0 The presence of two or more values indicates successive determinations for the developing stage, usually at 4%hour intervals. b Early 2nd stage is defined as the period within 5 days following molting, and late 2nd stage is 5-10 days following molting. c At this time the culture contained nearly equal numbers of 1st and 2nd stage larvae.
CATALASE
of any considerable amount of peroxidase activity. Some attempts to determine directly peroxidase activity in homogenates with guaiacol were unsuccessful, which further indicated the absence of any considerable peroxidase activity. Although catalase activity could be demonstrated by the three methods employed, only the titrimetric assay resulted in accurate reproducibility of specific activities of different preparations. The relationship of catalase activity, cytochrome c oxidase activity, and respiration of embryonating Ascaris eggs is presented in Fig. 4. A comparison of the theoretical amount of HzOz produced from the oxygen consumption and the decomposition of HzOz by catalase reveals a great excess of catalase activity throughout em-
% Q 210 2 5
-200
227
IN ASCARZS EGGS
bryonation. This relationship is of particular significance in fertilized unembryonated eggs in which cytochrome c oxidase activity was not demonstrable (1, 2). In the previous reports (1, 2) the authors suggested the presence of a flavin oxidase resulting in the formation of Hz02 to account for the oxygen consumption of unembryonated eggs. The presence of catalase in these preparations could certainly serve the function of decomposing any HzOz formed as a result of respiration. Of further interest is the fact that catalase activity remains constant through cleavage and gastrulation although marked changes in respiration and cytochrome c oxidase activity occur during this period (Fig. 4). Since the presence of cytochrome oxidase could account for respiration (2), the possible role of catalase is
=---OXYGEN CONSUMPTION -GYTOGHROYE OXIDASE ACTIVITY """'"""CATALASE ACTIVITY I
lI(III
III I STAGE
=
I 0
composite changes in catalase activity during development and its comparison with the cytochrome c oxidase activity and respiration of embryonating Ascuris eggs. The curve for cytochrome c oxidase (specific activity is mrmoles X lO/hour/mg. protein) is taken from Oya et al. (2). The curve for respiration is essentially from the results of Passey and Fairbairn (11). Catalase activity is expressed as pmoles Hz02 decomposed/minute/mg protein. The stages of development have been divided as follows: I, l-cell to 16-32 cell; II, 16-32 cell to gastrula; III, gastrula to developed 1st stage larval; IV, 2nd stage larval.
228
COSTELLO
difficult to relate. Possibly part of the respiration continues to be mediated via flavin oxidase and the catalase activity insures the decomposition of any HzOz produced. The decline in catalase activity (1st stage larval egg) appears to be associated with the decrease in respiration during this period which would reduce the amount of Hz02 capable of being produced. In 2nd stage larval eggs, the increased catalase as well as cytochrome c oxidase activity is not associated with the concurrent respiration. Such changes appear to be related to some future activity of the larvae, possibly infectivity. As suggested earlier (Z), the high cytochrome oxidase activity indicated that respiration and energy production during infectivity probably depends upon the cytochrome system. Perhaps the increased catalase activity is a protective measure against possible H202 formation at a time when respiration might be elevated or Hz02 formation by the host. At present one can only speculate in regard to these relationships. Although a marked increase in both catalase and cytochrome c oxidase activities is apparent in initial 2nd stage larvae (immediately following molting), the changes in activity of these enzymes differ during the following lo-day period (Fig. 4). Catalase activity remains relatively constant as contrasted with the pronounced increase in cytochrome oxidase activity. These results would indicate that the increase in the latter was not due to the number of 1st stage larvae which had completed molting. Under such conditions, the changes in activities of both enzymes should be parallel. Microscopic examination of the larvae confirms this conclusion in that 75% or more of the 1st stage larvae molted within an initial $-day period in these cultures. The presence of high catalase activity in Ascaris eggs is unlike the condition reported in adults (9). While the developing eggs seem to be adequately protected against peroxide toxicity, the adults apparently are
AND
SMITH
quite susceptible to HzOz. In developing sea urchin eggs, Deutsch and Gustafson (10) demonstrated that catalase activity was constant during cleavage followed by a marked decrease in mesenchymeblastula stage remaining relatively low through gastrulation and development of the larval stage. The authors attributed the decreased activity to the possible formation of an inhibitor. However, no increased activity was observed in sea urchin eggs corresponding to that in Ascaris eggs. This would further suggest that the change in catalase activity as well as cytochrome oxidase in 2nd stage larval eggs is a biochemical adaptation associated with infectivity. ACKNOWLEDGMENT We thank the personnel of the SchluderbergKurdle Company of Baltimore for their kind cooperation and assistance in supplying the ascarids utilized in this investigation. REFERENCES 1.
COSTELLO,L. C., OYA, H., AND SMITH, W., Arch.
Biochem.
Biophys.
103, 345 (1963).
2. OYA, H., COSTELLO,L. C., AND SMITH, W., J. Cell. Comp. Physiol.
62, 287 (1963)
3. COSTELLO, L. C., Exptl. Parasitol. 16,l (1964). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDAL,R. T., J. Biol. Chem. 193, 265 (1951).
5. CHANCE,B., ANDMEAHLY,A.C.,in“Methods in Enzymology” (S. I?. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 764. Academic Press, New York, 1955. 6. MEAHLY,A.C., ANDCHANCE,B.,in“Methods of Biochemical Analysis” (D. Glick, ed.), Vol. I, p. 357. Wiley (Interscience), New York, 1954. 7. BEERS,R. F., JR., AND SIZER, I. W., J. Biol. Chem. 196, 133 (1952). 8. HERBERT,D., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 784. Academic Press, New York, 1955. 9. LASER,H., Biochem. J. 38, 333 (1944). T., Arkiv 10. DEUTSCH,H. F., AND GUSTAFSON, Kemi 4, 221 (1952). 11. PASSEY,R. F., AND FAIRBAIRN,D., Can. J. Biochem. Physiol. 33, 1033 (1955).
OF
BIOCHEMISTRY
AND
The Comparative
BIOPHYSICS
106, 223-228 (1964)
Biochemistry
V. Changes
of Developing
in Catalase
Activity
L. C. COSTELLO From the Department
AND
during
January
Eggs
Embryonation’
w. SMITH2
of Physiology, School of Pharmacy, Baltimore, Maryland Received
Ascaris
University
of Maryland,
28, 1964
Catalase activity was identified in homogenates of eggs of Ascaris lumbricoides var. suum. The specific activity of catalase (pmoles Hz02 decomposed per minute per milligram protein) was 30, 16, and 54 in the l-cell, 1st stage larval, and 2nd stage larval eggs, respectively. The initial catalase activity observed in fertilized unembryonated eggs (l-cell) remained constant through cleavage and gastrulation until development of the 1st stage larvae, at which time activity markedly decreased (50%). When 1st stage larvae molted to 2nd stage larvae, an immediate and marked increase (over 300yc) in activity resulted. The relationship of catalase, cytochrome oxidase, and respiration in embryonating eggs is discussed. Catalase activity through all stages of embryonation was in great excess of theoretically formed H202, apparently serving as a protective mechanism against peroxide toxicity.
In previous studies (1, Z), changes in the terminal electron transport system during embryonation of Ascaris eggs were demonstrable. In initial unembryonated eggs (fertilized uterine eggs), the authors proposed that oxygen consumption was mediated in the absence of sufficient cytochrome c oxidase activity possibly via flavin oxidase. This proposed pathway would result in the formation of H20z and would require the presence of a mechanism for the removal of peroxide. The present report provides evidence for the presence of eatalase activity in initial unembryonated eggs and a comparison of the activity during embryonation. MATERIALS
AND
METHODS
Fertilized unembryonated eggs were harvested and cultured as previously described (1, 3). Washed eggs were suspended in potassium phos1 This investigation was supported by NSF research grant G-23313 and by NASA grant NsG436. 2 Predoctoral Trainee, P.H.S. grant DT-46, in conjunction with the Department of Histology and Embryology, School of Dentistry. 223
phate buffer (0.05 M, pH 7.0) and homogenized with a French Pressure Cell under a force of 16,000 pounds. The homogenate was centrifuged at 4009 for 2 minutes and the resulting supernatant fluid (experimental homogenate) passed through glass wool. Such preparations contained 10-20 mg protein per milliliter. Immediately prior to the assay, the homogenate was further diluted (1:lO) with phosphate buffer (0.01 M, pH 7.0). All steps were performed in the cold. Protein was determined by the method of Lowry et al. (4). The manometric determination of catalase activity was performed by conventional Warburg techniques (5). Single-sidearm flasks (approx. 17 ml capacity) were utilized, and the gas phase was air. The complete system generally contained 1.0 ml homogenate, 2.1 ml phosphate buffer (0.01 M, pH 7.0) and 0.1 ml H202 (0.2 M). The temperature was 18” or 28°C. and the shaking rate was 100 strokes per minute. After 8 minutes equilibration, the H202 was tipped into the main compartment, and the oxygen that evolved was measured. The spectrophotometric assay of catalase activity was performed by “tracking” the decomposition of Hz02 at 230 mp (6,7). Assays were performed in l-cm cuvettes with a Hitashi/PerkinElmer spectrophotometer. Generally, the reaction system contained 0.3 ml homogenate, 20 pmoles
224
COSTELLO
HzOz, and phosphate buffer (0.01 M, pH 7.4) to adjust the volume to 3.0 ml. The peroxide was added with a blow-out pipette immediately prior to tracking. The blank contained homogenate and buffer. The iodine-titration assay of catalase activity was performed as described by Herbert (8). The reactions were conducted in test tubes (6 X 1 inch). The complete system contained 5.0 ml HIOz (0.02 M prepared in 0.01 M phosphate buffer, pH 7.0) and 1.0 ml homogenate. The reaction was terminated at various intervals by the addition of 2.0 ml 1 N HzS04. The remaining Hz02 was determined by titrating the entire contents of each tube in situ with thiosulfate as described (8). RESULTS
The preliminary identification of catalase activity in homogenates of eggs was established by manometric techniques. Figure 1 demonstrates the evolution of 02 from HzOz in the presence of unembryonated egg homogenate. With heated homogenate (5 minutes in a boiling water bath) no gas production was observed. The addition of cyanide (5.0 pmoles) completely inhibited the activity. In the absence of HzOz, no gas production by homogenate was detected. The results demonstrated the catalatic effect of homogenates. Furthermore, the reaction rates at 18” and 28” demonstrated 2107
0
o TEMC! 28OC
I2
3 4 5 6 7 8 910 TIME (Minutes)
FIG. 1. The evolution of oxygen from hydrogen peroxide by unembryonated egg homogenate. The system contained 20 pmoles Hz02.
AND SMITH
only a slight increase in activity with temperature which is similar to the properties of catalase (7). Of particular significance was the stoichiometry of the reaction. The 9.5 pmoles O2 evolved accounted for essentially all of the Hz02 (20 pmoles) added to the system. The decomposition of HzOz by unembryonated egg homogenates was apparently the result of catalase activity with little or no peroxidatic activity involved. Identical conditions and properties were observed when homogenates of embryonated eggs were employed. However, this assay procedure was inadequate for the determination of specific activities of catalase. Under identical conditions manometrically determined activities were approximately 16 % of titrimetrically established values. Such limitations of gasometric procedures for catalase have been described and reviewed by others (6). The spectrophotometric determination of catalase activity in homogenates of unembryonated eggs is demonstrated in Fig. 2. Because of the high activity the disappearance of HzOz could be assayed directly in these preparations. The rate of decomposition of Hz02 was stimulated by increasing the concentration of homogenate or substrate. The reaction was inhibited by adding cyanide or aside to the system or by heating the homogenate (5 minutes in a boiling water bath). While the spectrophotometric assay successfully provided further evidence of catalase activity, the procedure was limited by the turbidity of the homogenate. For example, more concentrated homogenates were too turbid for assay, and lower concentrations did not provide adequate activity for measurement. Accurate and reproducible specific activities under these conditions could not be determined, particularly with different homogenate preparations. The results of a typical titrimetric assay of catalase activity in egg homogenates are presented in Fig. 3. Catalase activity was highest during the initial l&second interval. Furthermore, some assays were conducted with lo-second intervals which also resulted in decreasing activity with time. Because of the difficulty in manipulation for lo-
IN ASCARIS
CATALASE
.I2 .I I
225
EGGS
o ~O/JMOLES l IO~MOLES X 30pMOLES
o 0.4 I’ll1 HOMOGENATE l 0.3 ITli. HOMOGENATE X 0.2 t-tll. HOMOGENATE
/
.I0
/x
I
0
I
I
I
I
I
I
I
I
I
I
I
20 40 60 80 100 120 140
20 40 60 80 100 I20
TIME (Seconds) FIG. 2. The spectrophotometric determination of the decomposition of hydrogen peroxide by unembryonated egg homogenate. On the left the concentration of homogenate was varied, and the system contained 20 pmoles of hydrogen peroxide. On the right the concentration of hydrogen peroxide was varied, and 0.3 ml homogenate was used. The homogenate preparations contained 1.82 mg. protein per milliliter homogenate.
401
35 30 25 20i
X ONE-CELL EGGS 0 IST STAGE LARVAL EGGS /" X
/
X
/
X
second readings, subsequent assays were performed by using 15second intervals. The adoption of this titrimetric procedure for determination of catalase activity of homogenate provided excellent reproducibility. In all experiments, the systems were prepared in triplicate and the repeated titration values of thiosulfate agreed within 0.1 ml. Furthermore, the homogenate preparations did not interfere with the iodine titration nor result in any “blank” H202 values. The catalase activity as determined titrimetrically was inhibited by heating the f
0
I5 30 45 60 75 TIME (Seconds)
FIG. 3. The titrimetric assay of catalase activity in homogenates of l-cell eggs and 1st stage larval eggs. The system contained 100 rmoles hydrogen peroxide and 1.0 ml homogenate in a final volume of 6.0 ml. The homogenate of l-cell eggs contained 1.44 mg protein per milliliter homogenate and the 1st stage larval preparation contained 0.94 mg protein per milliliter homogenate.
226
COSTELLO
AND
homogenate or by adding cyanide or azide. Having established these relationships, the specific activity (pmoles HzOz decomposed per minute per milligram protein) of homogenate preparations was determined from the initial 15second interval by using 1.0 ml diluted homogenate (containing 1.0-2.0 mg protein) and 100 ctmoles of HzOz. Under these conditions activity was essentially proportional (within 10 %) to homogenate concentration. The catalase activities of some different homogenate preparations are presented in Table I. The results demonstrated significant differences in fertilized unembryonated eggs (l-cell stage), 1st stage larval, and 2nd TABLE THE
OF
Specific activity (,~nwles H20~ decomposed/ minute/milligram protein)
Observation No.~
Mean
stage larval eggs. The 1st stage larval eggs exhibited only 50% of the activity observed initially, whereas activity was markedly increased in eggs containing 2nd stage larvae. The means and standard deviations demonstrated the excellent reproducibility in different preparations. Having demonstrated these significant changes in catalase activity of l-cell, 1st stage larval, and 2nd stage larval eggs, investigations were conducted to determine the activity during embryonation (Table II). The initial catalase activity remained relatively constant through cleavage to the tadpole stage. However, activity rapidly declined (50 %) at the time of development of the 1st stage larvae and remained constant throughout this stage. Immediately following molting, the activity increased (200 %) in the 2nd stage larvae.
I
CATALASE ACTIVITY OF HOMOGENATES DIFFERENT STAGES OF ASCARIS EGGS
1 2 3 4 5 6 =k SD
DISCUSSION
l-Cd state stage
1st Stage Ianal eggs
2nd Stage larval eggs
25.6 32.0 25.6 32.0 30.6 33.2 29.8 f3.4
14.0 16.0 16.4 18.0 15.0 15.8 f1.6
52.0 54.0 57.2 60.0 46.8 54.0 f5.2
= Each observation represents tivity of a different preparation.
the catalase
The properties of catalase and the difficulties and conditions for its assay in various types of preparations have been recently and extensively reviewed (6). Due to the use of crude preparations in the present report, catalase activity was determined by three different assay procedures. The successful incorporation of the manometric, spectrophotometric, and titrimetric assays demonstrated the presence of catalase in Ascaris egg preparations. Furthermore, the stoichiometric results obtained by the manometric assays indicated the absence
ac-
TABLE CHANGES
IN
CATALASE ACTIVITY Specific
Culture
SMITH
l-Cell
2-4 Cell
activity
(qnoles
Gastrula-
$$f;
II
DURING EMBRYONATION
tadpole
1
32.0
26.0
-
-
2 3
30.6 32.0
28.4
24.0
32.0 26.2
4 5
-
-
28.8
27.2
Hz02
OF ASCARIS EGGS
decomposed/minute/milligram
protein)
2nd Stage larval0 eggs 1st stage
larvala
eggs Early
14.0 15.0,
17.6
b
Late
52.0
70.0,
-
46.8 -
16.4, 16.4, 18.0, 32.0” 13.2
57.2 -
60.0,
64.2 60.0 -
0 The presence of two or more values indicates successive determinations for the developing stage, usually at 4%hour intervals. b Early 2nd stage is defined as the period within 5 days following molting, and late 2nd stage is 5-10 days following molting. c At this time the culture contained nearly equal numbers of 1st and 2nd stage larvae.
CATALASE
of any considerable amount of peroxidase activity. Some attempts to determine directly peroxidase activity in homogenates with guaiacol were unsuccessful, which further indicated the absence of any considerable peroxidase activity. Although catalase activity could be demonstrated by the three methods employed, only the titrimetric assay resulted in accurate reproducibility of specific activities of different preparations. The relationship of catalase activity, cytochrome c oxidase activity, and respiration of embryonating Ascaris eggs is presented in Fig. 4. A comparison of the theoretical amount of HzOz produced from the oxygen consumption and the decomposition of HzOz by catalase reveals a great excess of catalase activity throughout em-
% Q 210 2 5
-200
227
IN ASCARZS EGGS
bryonation. This relationship is of particular significance in fertilized unembryonated eggs in which cytochrome c oxidase activity was not demonstrable (1, 2). In the previous reports (1, 2) the authors suggested the presence of a flavin oxidase resulting in the formation of Hz02 to account for the oxygen consumption of unembryonated eggs. The presence of catalase in these preparations could certainly serve the function of decomposing any HzOz formed as a result of respiration. Of further interest is the fact that catalase activity remains constant through cleavage and gastrulation although marked changes in respiration and cytochrome c oxidase activity occur during this period (Fig. 4). Since the presence of cytochrome oxidase could account for respiration (2), the possible role of catalase is
=---OXYGEN CONSUMPTION -GYTOGHROYE OXIDASE ACTIVITY """'"""CATALASE ACTIVITY I
lI(III
III I STAGE
=
I 0
composite changes in catalase activity during development and its comparison with the cytochrome c oxidase activity and respiration of embryonating Ascuris eggs. The curve for cytochrome c oxidase (specific activity is mrmoles X lO/hour/mg. protein) is taken from Oya et al. (2). The curve for respiration is essentially from the results of Passey and Fairbairn (11). Catalase activity is expressed as pmoles Hz02 decomposed/minute/mg protein. The stages of development have been divided as follows: I, l-cell to 16-32 cell; II, 16-32 cell to gastrula; III, gastrula to developed 1st stage larval; IV, 2nd stage larval.
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difficult to relate. Possibly part of the respiration continues to be mediated via flavin oxidase and the catalase activity insures the decomposition of any HzOz produced. The decline in catalase activity (1st stage larval egg) appears to be associated with the decrease in respiration during this period which would reduce the amount of Hz02 capable of being produced. In 2nd stage larval eggs, the increased catalase as well as cytochrome c oxidase activity is not associated with the concurrent respiration. Such changes appear to be related to some future activity of the larvae, possibly infectivity. As suggested earlier (Z), the high cytochrome oxidase activity indicated that respiration and energy production during infectivity probably depends upon the cytochrome system. Perhaps the increased catalase activity is a protective measure against possible H202 formation at a time when respiration might be elevated or Hz02 formation by the host. At present one can only speculate in regard to these relationships. Although a marked increase in both catalase and cytochrome c oxidase activities is apparent in initial 2nd stage larvae (immediately following molting), the changes in activity of these enzymes differ during the following lo-day period (Fig. 4). Catalase activity remains relatively constant as contrasted with the pronounced increase in cytochrome oxidase activity. These results would indicate that the increase in the latter was not due to the number of 1st stage larvae which had completed molting. Under such conditions, the changes in activities of both enzymes should be parallel. Microscopic examination of the larvae confirms this conclusion in that 75% or more of the 1st stage larvae molted within an initial $-day period in these cultures. The presence of high catalase activity in Ascaris eggs is unlike the condition reported in adults (9). While the developing eggs seem to be adequately protected against peroxide toxicity, the adults apparently are
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quite susceptible to HzOz. In developing sea urchin eggs, Deutsch and Gustafson (10) demonstrated that catalase activity was constant during cleavage followed by a marked decrease in mesenchymeblastula stage remaining relatively low through gastrulation and development of the larval stage. The authors attributed the decreased activity to the possible formation of an inhibitor. However, no increased activity was observed in sea urchin eggs corresponding to that in Ascaris eggs. This would further suggest that the change in catalase activity as well as cytochrome oxidase in 2nd stage larval eggs is a biochemical adaptation associated with infectivity. ACKNOWLEDGMENT We thank the personnel of the SchluderbergKurdle Company of Baltimore for their kind cooperation and assistance in supplying the ascarids utilized in this investigation. REFERENCES 1.
COSTELLO,L. C., OYA, H., AND SMITH, W., Arch.
Biochem.
Biophys.
103, 345 (1963).
2. OYA, H., COSTELLO,L. C., AND SMITH, W., J. Cell. Comp. Physiol.
62, 287 (1963)
3. COSTELLO, L. C., Exptl. Parasitol. 16,l (1964). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDAL,R. T., J. Biol. Chem. 193, 265 (1951).
5. CHANCE,B., ANDMEAHLY,A.C.,in“Methods in Enzymology” (S. I?. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 764. Academic Press, New York, 1955. 6. MEAHLY,A.C., ANDCHANCE,B.,in“Methods of Biochemical Analysis” (D. Glick, ed.), Vol. I, p. 357. Wiley (Interscience), New York, 1954. 7. BEERS,R. F., JR., AND SIZER, I. W., J. Biol. Chem. 196, 133 (1952). 8. HERBERT,D., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 784. Academic Press, New York, 1955. 9. LASER,H., Biochem. J. 38, 333 (1944). T., Arkiv 10. DEUTSCH,H. F., AND GUSTAFSON, Kemi 4, 221 (1952). 11. PASSEY,R. F., AND FAIRBAIRN,D., Can. J. Biochem. Physiol. 33, 1033 (1955).