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Peroxidase activity in platyhelminth cuticular mitochondria
EXPERIMENTAL
PARASITOLOGY
Peroxidase
23,
51-55
Activity
(1968)
in Platyhelminth Alvin
Department
of
Biology, California (Submitted
for
Cuticular
Mitochondrial
H. Rothman State College, Fullerton,
publication,
10 October
California 92631
1967)
ROTHMAN, ALVIN H. ( 1968). Peroxidase activity in platyhelminth cuticular mitochondria. Experimental Parasitology 23, 5155. Using diaminobenzidine and hydrogen peroxide, peroxidase activity was demonstrated in the cuticular mitochondria of the cestode Hymenokpis citelli and the trematode Haematoloechus medioplexus. This is believed to be the first electron microscopic demonstration of peroxidase activity in animal mitochondria. INDEX DESCRIPTORS: Peroxidase activity, in mitochondria; Hymenolepis cite& mitochondria of: Haematdoechus mediozllexus, mitochondria of; mitochondria, peroxidase activity in; Enzyme; Peroxidase.
An attempt to detect protein transport in a cestode, Hymenolepk cite&, and in a trematode, Haematoloechus medioplexus, using purified horseradish peroxidase (as suggested by the work of Becker, Novikoff, and Zimmerman, 1967; Graham and Karnovsky, 1965, 1966) led to unexpected observations ( Rothman, unpublished), The cuticular mitochondria in both experimental groups of animals showed a positive reaction to the peroxidase test, whereas no reaction product was found elsewhere in the cuticle. The present report covers further tests of the peroxidase reaction in the cuticular mitochondria of HymenoZepis and Haematoloechus. MATERIALS
AND
METHODS
For each experiment a Syrian hamster containing the strobilar form of Hymenolepi.s citelli was killed by a blow on the head and the cestodes were flushed from the small intestine with Kreb’s Ringer Tris maleate solution (KRTM), pH 7.4. Also, for each experiment a grass frog (in some cases two or three in order to find the re‘This work was supported by a grant, 06442, from the National Institutes of Health.
AI 51
quired number of worms for each experiment) was killed by pithing the brain and spinal cord and the Haematoloechus medioplexus removed from the lungs in KRTM, pH 7.4. The worms were rinsed three times in KRTM-care being taken to remove all debris-and fixed for 1 hour in ice-cold 3% glutaraldehyde prepared in 0.1 M cacodylate buffer at pH 7.4, followed by six lo-minute rinses in cold buffer. Sections, 250~ thick, were prepared on a Smith and Farquahar tissue sectioner and used in one of the test solutions (suggested by Graham and Karnovsky, lQ66) which consisted of either .05 M Tris-HCl buffer, pH 7.4; .05% 3,3-diaminobenzidine tetrahydrochloride (DAB) (K and K Laboratories) dissolved in the Tris-HCl buffer; .Ol% H,O, in the Tris-HCl buffer; or the complete test medium consisting of .O% DAB and .01X H,O, in the Tris-HCl buffer. Enzyme inhibition was accomplished by: First, the inclusion of .Ol M sodium cyanide in the complete test media, and second, using sections that were heated in boiling water for 30 minutes prior to incubation in the complete test solution. The sections were incubated for 10 minutes, rinsed three times with dis-
52
F 'IG. 1. Untreated mitt jchondria.
ROTHMAN
section
of Hymenolepis
F ‘IG. 2. Section of Hymenolepis heah ‘y precipitate in mitochondria. :le bundle in parenchyma.
citelli
cuticle
showing
a mitochondrion.
X19,000.
cite& treated with diaminobenzidine and hydrogen peroxide sho wing X10,000. C = cuticle; m = mitochondria; SC = subcuticle; I?=
PEROXIDASE,
FIG. 3. Untreated section mitochondria; CS = cuticular
of Haematoloechus spine.
CUTICLE
medioplexus
FIG. 4. Section of Haematoloechus medioplexus treated ide showing precipitate in some mitochondria (arrow). X25,000.
53
MITOCHONDRIA
showing
a mitochondrion.
X12,000.
m =
with diaminobenzidine and hydrogen peroxX18,000. Insert: Precipitate on mitochondria.
54
ROTHMAN
tilled water, fixed for 1 hour with 1% OSmium tetroxide in 0.1 M cacodylate bufferpH 7.4, dehydrated through an ethyl alcohol series (25% 50%, 70% 95%, and 100%) followed by propylene oxide, propylene oxide in Maraglas (l:l), and then embedded in Maraglas. A 2% solution of uranyl acetate in 70% ethanol was incorporated in the dehydration procedure. Three experiments were done utilizing portions of at least two worms of each type in each test medium. At least one cuticular region of each worm from each test medium was examined.
membranes. When DAB or H,O, was omitted from the complete test solution, no precipitate formed in the gross sections or on the mitochondria. The .Ol M cyanide completely inhibited the color change and the formation of mitochondrial precipitate. The prior heating of the 250-p sectioned worms effected a reduction of the precipitate as evidenced by the light-brown color of the sections. The mitochondria (Fig. 5) are poorly preserved after heating and the precipitate is less than in the unheated experimental samples, but more than in the control sections (Figs. 1 and 3). DISCUSSION
RESULTS The gross 250-+L worm sections in the complete medium became brown during the incubation period. All mitochondria in the cestode cuticle (Fig. 2) and some mitochondria in the trematode cuticle (Fig. 4) had reaction product deposited on their
I TIG. 5. Section of heat treated experimental part :d to the unheated
Hynwnolepis worms.
citelli
X12,000.
Wachstein and Meisel (1964) in a report on a light microscope investigation of peroxidase distribution in several mammalian tissues (leukocytes, kidney, liver, salivary gland, thyroid gland, pancreas, skin, skeletal muscle, and heart), suggest that some of the peroxidase activity of only
showing a reduced m = mitochondria.
quantity
of precipitate
(
PEROXIDASE,
CUTICLE
the skeletal muscle and heart occurs at sites considered to be mitochondria. The present author has found no other reports on mitochondrial peroxidase activity. The general absence of mitochondrial peroxidase activity in mammalian tissue is supported by the electron microscope photomicrographs of Becker et al. (1967) working with choroid plexus; and Graham and Karnovsky (1965, 1966) working with kidney, which show electron micrographs of mitochondria lacking precipitate, whereas peroxidase-generated precipitate is seen elsewhere in the sections. Therefore, the localization of platyhelminth peroxidase varies from the localization of other animal peroxidases already studied in that the mitochondria of flatworms contain peroxidase activity. Cheah (1967a) demonstrated peroxidase activity in the cestode Moniexia expansa, and also showed (Cheah, 1967b) that M. expansa forms hydrogen peroxide. Cheah’s studies were primarily of a biochemical nature and the peroxidase was not localized. A brief concluding statement without detail, in a paper on cytochrome oxidase, by Seligman et al. (1967) suggests that cytochrome oxidase may be located in mitochondria by DAB. (Seligman indicates that a publication on the subject is in preparation. ) Read (1952) demonstrated cytochrome oxidase activity in homogenates of Hymenolepis diminutu and Cheah (196713) demonstrated cytochrome oxidase activity in a mitochondrial preparation from M. expansa. Further information, therefore, on Seligman’s method of demonstrating cytochrome oxidase with DAB will permit an explanation of the nonreaction of cytochrome oxidase with DAB in flatworms. Moreover, the other works mentioned above, using DAB, contrary to Seligman’s ( 1967) suggestion, report no precipitate formation in the tissue mitochondria-
MITOCHONDRIA
55
which are known to contain cytochrome oxidase. The present report that no precipitate forms on the mitochondria of Hymenolepis and Haematoloechus, when H,O, is omitted and DAB is present in the test media, infers that peroxidase and not another oxidase is involved in the formation of the oxidized DAB precipitate. ACKNOWLEDGMENT The author thanks Mrs. Janice for her fine technical assistance.
Edwards-Elders
REFERENCES BECXJXR, N. H., NOVIKOFF, A. H., AND Zr~r+mnMAN, H. M. 1967. Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus. ]ournuZ of Histochemistry and Cytochemistq 15, 160165. CHEAH, K. S. 1967a. Histochemical and spectro-
photometric demonstrationof peroxidasein Monfezia expansa ( Cestoda ) . Comparative Biochemistry CHEAH,
K.
S.
and Physiology 1967b.
Moniezia ezpansa Biochemistry and Physiology GRAHAM,
21, 351-355.
The oxidase (Cestoda).
R. C., AND KARNOVSKY,
systems Comparative
of
23, 277302. M.
J. 1965.
The
early stagesof absorptionof injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. Journal of Histochemistry and Cytochemistry 14, 291302. GRAHAM, R. C., AND KARNOVSKY, M. J. 1966. Glomerular permeability. Ultrastructural cytochemical studies using peroxidases as protein tracers. Journal of Experimental Medicine 124, 1123-1134. READ, C. P. 1952. Contributions to c&ode enzymology I. The cytochrome system and succinic dehydrogenase in Hymenolepis diminuta. Experimental Parasitology 1, 353-362. SELIGMAN, A. M., PLAPINGER, R. E., WASSERKRUG, H. L., DEB, C., AND HANKER, J. S. 1967. Ultrastructural demonstration of cytochrome oxidase activity by the Nadi reaction with osmiophilic reagents. The Journal of Cell
Biology 34, 787-800. WACHSTEIN, stration
M., AND MELSEL, E. of peroxidase activity
1964. Demonin tissue sec-
tions. journal of Histochemistry chemisty 12, 536-544.
and Cyto-
PARASITOLOGY
Peroxidase
23,
51-55
Activity
(1968)
in Platyhelminth Alvin
Department
of
Biology, California (Submitted
for
Cuticular
Mitochondrial
H. Rothman State College, Fullerton,
publication,
10 October
California 92631
1967)
ROTHMAN, ALVIN H. ( 1968). Peroxidase activity in platyhelminth cuticular mitochondria. Experimental Parasitology 23, 5155. Using diaminobenzidine and hydrogen peroxide, peroxidase activity was demonstrated in the cuticular mitochondria of the cestode Hymenokpis citelli and the trematode Haematoloechus medioplexus. This is believed to be the first electron microscopic demonstration of peroxidase activity in animal mitochondria. INDEX DESCRIPTORS: Peroxidase activity, in mitochondria; Hymenolepis cite& mitochondria of: Haematdoechus mediozllexus, mitochondria of; mitochondria, peroxidase activity in; Enzyme; Peroxidase.
An attempt to detect protein transport in a cestode, Hymenolepk cite&, and in a trematode, Haematoloechus medioplexus, using purified horseradish peroxidase (as suggested by the work of Becker, Novikoff, and Zimmerman, 1967; Graham and Karnovsky, 1965, 1966) led to unexpected observations ( Rothman, unpublished), The cuticular mitochondria in both experimental groups of animals showed a positive reaction to the peroxidase test, whereas no reaction product was found elsewhere in the cuticle. The present report covers further tests of the peroxidase reaction in the cuticular mitochondria of HymenoZepis and Haematoloechus. MATERIALS
AND
METHODS
For each experiment a Syrian hamster containing the strobilar form of Hymenolepi.s citelli was killed by a blow on the head and the cestodes were flushed from the small intestine with Kreb’s Ringer Tris maleate solution (KRTM), pH 7.4. Also, for each experiment a grass frog (in some cases two or three in order to find the re‘This work was supported by a grant, 06442, from the National Institutes of Health.
AI 51
quired number of worms for each experiment) was killed by pithing the brain and spinal cord and the Haematoloechus medioplexus removed from the lungs in KRTM, pH 7.4. The worms were rinsed three times in KRTM-care being taken to remove all debris-and fixed for 1 hour in ice-cold 3% glutaraldehyde prepared in 0.1 M cacodylate buffer at pH 7.4, followed by six lo-minute rinses in cold buffer. Sections, 250~ thick, were prepared on a Smith and Farquahar tissue sectioner and used in one of the test solutions (suggested by Graham and Karnovsky, lQ66) which consisted of either .05 M Tris-HCl buffer, pH 7.4; .05% 3,3-diaminobenzidine tetrahydrochloride (DAB) (K and K Laboratories) dissolved in the Tris-HCl buffer; .Ol% H,O, in the Tris-HCl buffer; or the complete test medium consisting of .O% DAB and .01X H,O, in the Tris-HCl buffer. Enzyme inhibition was accomplished by: First, the inclusion of .Ol M sodium cyanide in the complete test media, and second, using sections that were heated in boiling water for 30 minutes prior to incubation in the complete test solution. The sections were incubated for 10 minutes, rinsed three times with dis-
52
F 'IG. 1. Untreated mitt jchondria.
ROTHMAN
section
of Hymenolepis
F ‘IG. 2. Section of Hymenolepis heah ‘y precipitate in mitochondria. :le bundle in parenchyma.
citelli
cuticle
showing
a mitochondrion.
X19,000.
cite& treated with diaminobenzidine and hydrogen peroxide sho wing X10,000. C = cuticle; m = mitochondria; SC = subcuticle; I?=
PEROXIDASE,
FIG. 3. Untreated section mitochondria; CS = cuticular
of Haematoloechus spine.
CUTICLE
medioplexus
FIG. 4. Section of Haematoloechus medioplexus treated ide showing precipitate in some mitochondria (arrow). X25,000.
53
MITOCHONDRIA
showing
a mitochondrion.
X12,000.
m =
with diaminobenzidine and hydrogen peroxX18,000. Insert: Precipitate on mitochondria.
54
ROTHMAN
tilled water, fixed for 1 hour with 1% OSmium tetroxide in 0.1 M cacodylate bufferpH 7.4, dehydrated through an ethyl alcohol series (25% 50%, 70% 95%, and 100%) followed by propylene oxide, propylene oxide in Maraglas (l:l), and then embedded in Maraglas. A 2% solution of uranyl acetate in 70% ethanol was incorporated in the dehydration procedure. Three experiments were done utilizing portions of at least two worms of each type in each test medium. At least one cuticular region of each worm from each test medium was examined.
membranes. When DAB or H,O, was omitted from the complete test solution, no precipitate formed in the gross sections or on the mitochondria. The .Ol M cyanide completely inhibited the color change and the formation of mitochondrial precipitate. The prior heating of the 250-p sectioned worms effected a reduction of the precipitate as evidenced by the light-brown color of the sections. The mitochondria (Fig. 5) are poorly preserved after heating and the precipitate is less than in the unheated experimental samples, but more than in the control sections (Figs. 1 and 3). DISCUSSION
RESULTS The gross 250-+L worm sections in the complete medium became brown during the incubation period. All mitochondria in the cestode cuticle (Fig. 2) and some mitochondria in the trematode cuticle (Fig. 4) had reaction product deposited on their
I TIG. 5. Section of heat treated experimental part :d to the unheated
Hynwnolepis worms.
citelli
X12,000.
Wachstein and Meisel (1964) in a report on a light microscope investigation of peroxidase distribution in several mammalian tissues (leukocytes, kidney, liver, salivary gland, thyroid gland, pancreas, skin, skeletal muscle, and heart), suggest that some of the peroxidase activity of only
showing a reduced m = mitochondria.
quantity
of precipitate
(
PEROXIDASE,
CUTICLE
the skeletal muscle and heart occurs at sites considered to be mitochondria. The present author has found no other reports on mitochondrial peroxidase activity. The general absence of mitochondrial peroxidase activity in mammalian tissue is supported by the electron microscope photomicrographs of Becker et al. (1967) working with choroid plexus; and Graham and Karnovsky (1965, 1966) working with kidney, which show electron micrographs of mitochondria lacking precipitate, whereas peroxidase-generated precipitate is seen elsewhere in the sections. Therefore, the localization of platyhelminth peroxidase varies from the localization of other animal peroxidases already studied in that the mitochondria of flatworms contain peroxidase activity. Cheah (1967a) demonstrated peroxidase activity in the cestode Moniexia expansa, and also showed (Cheah, 1967b) that M. expansa forms hydrogen peroxide. Cheah’s studies were primarily of a biochemical nature and the peroxidase was not localized. A brief concluding statement without detail, in a paper on cytochrome oxidase, by Seligman et al. (1967) suggests that cytochrome oxidase may be located in mitochondria by DAB. (Seligman indicates that a publication on the subject is in preparation. ) Read (1952) demonstrated cytochrome oxidase activity in homogenates of Hymenolepis diminutu and Cheah (196713) demonstrated cytochrome oxidase activity in a mitochondrial preparation from M. expansa. Further information, therefore, on Seligman’s method of demonstrating cytochrome oxidase with DAB will permit an explanation of the nonreaction of cytochrome oxidase with DAB in flatworms. Moreover, the other works mentioned above, using DAB, contrary to Seligman’s ( 1967) suggestion, report no precipitate formation in the tissue mitochondria-
MITOCHONDRIA
55
which are known to contain cytochrome oxidase. The present report that no precipitate forms on the mitochondria of Hymenolepis and Haematoloechus, when H,O, is omitted and DAB is present in the test media, infers that peroxidase and not another oxidase is involved in the formation of the oxidized DAB precipitate. ACKNOWLEDGMENT The author thanks Mrs. Janice for her fine technical assistance.
Edwards-Elders
REFERENCES BECXJXR, N. H., NOVIKOFF, A. H., AND Zr~r+mnMAN, H. M. 1967. Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus. ]ournuZ of Histochemistry and Cytochemistq 15, 160165. CHEAH, K. S. 1967a. Histochemical and spectro-
photometric demonstrationof peroxidasein Monfezia expansa ( Cestoda ) . Comparative Biochemistry CHEAH,
K.
S.
and Physiology 1967b.
Moniezia ezpansa Biochemistry and Physiology GRAHAM,
21, 351-355.
The oxidase (Cestoda).
R. C., AND KARNOVSKY,
systems Comparative
of
23, 277302. M.
J. 1965.
The
early stagesof absorptionof injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. Journal of Histochemistry and Cytochemistry 14, 291302. GRAHAM, R. C., AND KARNOVSKY, M. J. 1966. Glomerular permeability. Ultrastructural cytochemical studies using peroxidases as protein tracers. Journal of Experimental Medicine 124, 1123-1134. READ, C. P. 1952. Contributions to c&ode enzymology I. The cytochrome system and succinic dehydrogenase in Hymenolepis diminuta. Experimental Parasitology 1, 353-362. SELIGMAN, A. M., PLAPINGER, R. E., WASSERKRUG, H. L., DEB, C., AND HANKER, J. S. 1967. Ultrastructural demonstration of cytochrome oxidase activity by the Nadi reaction with osmiophilic reagents. The Journal of Cell
Biology 34, 787-800. WACHSTEIN, stration
M., AND MELSEL, E. of peroxidase activity
1964. Demonin tissue sec-
tions. journal of Histochemistry chemisty 12, 536-544.
and Cyto-