INFECTION
AND
Vol. 12, No. 2 Printed in U.S.A.
IMMUNITY, Aug. 1975, p. 267-269
Copyright 0 1975 American Society for Microbiology
o-Diphenoloxidase of Mycobacterium leprae Separated from Infected Armadillo Tissues K. PRABHAKARAN,* E. B. HARRIS,
AND W.
F. KIRCHHEIMER
U.S. Public Health Service Hospital, Carville, Louisiana 70721
We reported earlier the occurrence of a unique o-diphenoloxidase in Mycobacterium leprae recovered from lepromatous human tissues. No other source of M. Ieprae for biochemical studies was available at the time. In the present report, properties of phenoloxidase in M. Ieprae separated from infected armadillo tissues are presented. The results show that the o-diphenoloxidase remains unaltered in the passage of the bacilli from the human to the animal host, indicating that the enzyme is an intrinsic characteristic of the leprosy bacteria.
The only information available in literature on the metabolic properties of Mycobacterium leprae is a series of studies we reported using concentrates of organisms separated from lepromatous human tissues (8-15). No authenticated culture of the bacillus is as yet available, and until recently no immunologically unimpaired animal model was known which, when inoculated with the leprosy bacteria, develops the heavy systemic form of the disease. Now it has been shown that M. Ieprae produces disseminated leprosy in the nine-banded armadillo (Dasypus novemcinctus Linn.) (2). We found that organisms separated from lepromatous human tissues contain an unusual form of the enzyme o-diphenoloxidase (o-diphenol:oxygen oxidoreductase [EC1.10.3.1]). The o-diphenoloxidase occurring in mammalian melanocytes oxidizes L-dopa (3,4-dihydroxyphenyalanine) to dopachrome (2-carboxy-2,3-dihydroindole-5,6quinone). Dopachrome is characterized by an absorbance maximum at 475 nm in the spectrum (5). The mammalian enzyme shows little activity towards D-dopa or derivatives of dopa like epinephrine and norepinephrine. The enzyme in M. leprae converted both D- and Ldopa to indole-5,6-quinone and also oxidized a variety of phenolic substrates to quinones (11). Indole-5,6-quinone is distinguished from dopachrome by its absorbance maximum at 540 nm (5). Since at the time of our earlier studies lepromatous human tissues were the only sources of M. leprae for biochemical studies, it was not possible to ascertain whether phenoloxidase is a constitutive enzyme in the bacillus or an enzyme induced by the milieu in which the organism proliferates. The present report deals with the o-diphenoloxidase of M. leprae separated from infected tissues of armadillos. The results show that the o-diphenoloxidase is a constitutive enzyme in M. leprae, which remains unaltered in the passage of bacilli from the human to the animal host.
MATERIALS AND METHODS
Organisms. Concentrates of M. leprae were separated from infected liver of armadillos. The tissues were collected aseptically and kept frozen at -20 or -80 C. The preparative procedure has been described earlier and is carried out at 0 to 2 C (8, 13). It involves homogenization of the tissue and differential and density gradient centrifugations of the homogenate in inert solutions, such as those of sucrose and KCI. The bacilli were disrupted by ultrasonic oscillation (12). Some preparations of the bacilli were treated with trypsin, acetone, ether, and dilute NaOH to remove any host tissue materials adsorbed superficially. These treated organisms also readily oxidized dopa. Liver tissue by itself did not oxidize either L- or D-dopa. In bacilli recovered from tissues of animals which had been dead for several hours, phenoloxidase was very low and rather inconsistent. Prolonged storage of the tissues at -20 or -80 C also resulted in considerable loss in the activity of the enzyme in the bacteria. Enzymes. Lyophilized mushroom tyrosinase (odiphenoloxidase) was purchased from Sigma Chemical Co. (St. Louis, Mo.) or from the ICN Life Sciences Group (Cleveland, Ohio). Cultured melanocytes served as a source of mammalian o-diphenoloxidase. The melanocyte cultures (derived from the Cloudman S-91 melanoma) were obtained from the American Type Culture Collection. The cells were grown at 37 C in Ham F-10 medium containing 10% fetal calf serum. Chemicals. D-Dopa was purchased from the ICN Life Sciences Group, and L-dopa was obtained from Sigma. DL- [G-3Hjdopa (labeling distributed generally, 250 mCi/mmol) was obtained from the Amersham-Searle Corp. (Arlington Heights, Ill.). Glass counting vials were purchased from Beckman Instruments, Inc. (Fullerton, Calif.), and the scintillation solution (Aquasol) was obtained from New England Nuclear Corp. (Boston, Mass.). Other chemicals used were those of the highest purity commercially available. Enzyme assay. Dopa is converted to quinone and water by o-diphenoloxidase, and oxygen is consumed in the process. When tritium-labeled dopa was used as substrate, activity of the labeled water formed was measured in a liquid scintillation counter. Details of the procedure have been described before (6, 12), 267
INFECT . IMMUN .
PRABHAKARAN, HARRIS, AND KIRCHHEIMER
268
which has been modified from the method originally reported by Pomerantz (7). The reaction system consisted of: Na,HPO4-KH,PO4 buffer (pH 6.8), 40 glmol; DL- [G-3H]dopa, 1 uCi; "cold" (unlabeled) L-dopa, 1 Mmol; M. leprae, 1.2 mg of protein; temperature, 37 C; time, 60 min; volume, 2 ml. Appropriate controls were run of heat-inactivated enzyme (100 C, 30 min) with substrate and of substrate without enzyme. Formation of quinone from dopa was assayed by determining the spectrum of the reaction mixture, as described before (11). The reaction system consisted of the following constituents in the final concentrations indicated: Na,HPO4-KH,P04 buffer (pH 6.8), 0.1 M; substrates (except epinephrine), 0.002 M; epinephrine, 0.0005 M; inhibitors, 0.004 M; M. leprae, 1.5 to 2.0 mg of protein; melanoma cells, 2 mg of protein; mushroom tyrosinase, 10 Ag; temperature, 37 C; time, 30 min; volume, 3 ml. We measured the absorbance maximum for dopachrome at 480 nm; it was identical to the value at 475 nm, since dopachrome gives a broad peak in the area. We have reported previously the uptake of oxygen by M. leprae in the oxidation of dopa (13). The effect of inhibitors on o-diphenoloxidase of M. leprae was assayed spectrophotometrically by comparing the absorbance maxima of the reaction products with and without the inhibitors (8).
RESULTS AND DISCUSSION Oxidation of substrates. The oxidation of substrates by o-diphenoloxidase from different sources is shown in Table 1. Mushroom tyrosin-
well as M. Ieprae recovered from armadillo tissues converted both D- and L-dopa to quinone at the same rate and also oxidized epinephrine and norepinephrine. These data are in agreement with those obtained with bacilli from human tissues (11). Mammalian tyrosinase oxidized L-dopa; however, it was inactive towards D-dopa and the catecholamines. Epinephrine undergoes rapid auto-oxidation at pH 6.8; to avoid this, the reaction was carried out at pH 6.5. Table 2 illustrates the oxidation of tritium-labeled dopa by M. leprae obtained from the armadillo. Heating the bacilli at 100 C for 30 min produced considerable loss of activity, indicating that the enzyme is labile, although some residual activity was still present. The effect of reducing agents on o-
diphenoloxidase from different sources is presented in Table 3. Reduced glutathione, ascorbic acid, and cysteine completely inhibited plant and mammalian tyrosinases. Reducing agents are known to be inhibitors of tyrosinase (3). However, these compounds showed little effect on the phenoloxidase of M. Ieprae. The bacilli used had been disrupted by ultrasonic oscillation. As such, the results observed are not due to the permeability barrier of the bacterial cell membrane. Apparently the active site of the enzyme in M. leprae is not readily accessible to the inhibitors, unlike in plant and mammalian tyrosinases. This phenomenon is further borne out by data on the effect of metal chelators on the enzyme from different sources, which are presented in Table 4. o-Diphenolkidase is known to be a copper protein (4). Cyanide is a potent inhibitor of metallo enzymes. Diethyldithiocarbamate (DDC) and pencillaTABLE 2. Oxidation of tritium-labeled dopa by M. leprae
a
ase as
['H Idopa
Bacilli
Total
oxidized (pmol)
Unheated Heated
132,159a 31,803
238.13 57.31
Values are corrected for auto-oxidation of dopa. TABLE 3. Effect of reducing agents on o-diphenoloxidase from different sources Reaction system
Enzyme + dopa Enzyme + dopa + ascorbate Enzyme + dopa + GSHa Enzyme + dopa + cysteine a
480 nm Absorbance Absorbance ateaoct at 540 nmMuhom (M. leprae) Mushroom Melanocyte tyrosinase
culture
0.108 0.098
0.120 0
0.140 0
0.080
0
0
0.095
0
0
GSH, Reduced glutathione.
* TABLE 1. Oxidation of dopa and its derivatives by M. leprae and by plant and mammalian tyrosinase: increase in absorbancea Substrate
M. leprae
Mushroom tyrosinase
Melanocyte
L-Dopa D-Dopa L-Epinephrine DL-Norepinephrine
0.100 (540)" 0.099 (540) 0.100 (480) 0.060 (480)
0.095 (480) 0.100 (480) 0.240 (480) 0.140 (480)
0.160 (480) 0 0 0
culture
a Absorbance values are corrected for absorbance of enzyme and nonenzymatic oxidation of substrates. h Values in parentheses indicate absorbance maxima in nanometers.
o-DIPHENOLOXIDASE IN ARMADILLO BACTERIA
VOL. 12, 1975
TABLE 4. Effect of metal chelators on o-diphenoloxidase from different sources Reaction system Enzyme + dopa Enzyme + dopa + NaCN Enzyme + dopa + penicillamine Enzyme + dopa + DDC
Absorbance Absorbance at 480 nm at 540 nm Mushroom Melanocyte (M. leprae) tyrosinase culture
0.099 0.095
0.130 0
0.140 0
0.100
0
0
0
0
0
mine are effective copper chelators, used in the treatment of hepatolenticular degeneration in man. All the three metal chelators produced total inhibition of mammalian and plant tyro-
269
The exact metabolic role of this unusual enzyme in the survival and proliferation of the leprosy organism still remains to be elucidated. ACKNOWLEDGMENTS This investigation was supported by grants from the U.S.-Japan Cooperative Medical Science Program of the National Institute of Allergy and Infectious Diseases (AI-07890 and AI-11204 BM) and by the World Health Organization.
LITERATURE CITD 1. Kirchheimer, W. F., and K. Prabhakaran. 1968. Metabolic and biologic tests on mycobacteria once labeled as leprosy bacilli. Int. J. Lepr. 36:162-165. 2. Kirchheimer, W. F., and E. E. Storrs. 1971. Attempts to establish the armadillo (Dasypus novemcinctus Linn.) as a model for the study of leprosy. I. Report of lepromatoid leprosy in an experimentally infected armadillo. Int. J. Lepr. 39:693-702. 3. Lemer, A. B., and T. B. Fitzpatrick. 1950. Bichemistry of melanin formation. Physiol. Rev. 30:91-126. 4. Makino, N., P. McMahill, and H. S. Mason. 1974. The oxidation state of copper in resting tyrosinase. J. Biol.
sinases. However, penicillamine and cyanide showed little effect on the o-diphenoloxidase activity of the bacilli. Probably, the configuraChenh 249:6062-6066. tion of the enzyme protein in M. leprae is such Structure of melanins, p. 563-582: that these inhibitors are unable to penetrate 5. Mason, H. S. 1959. In M. Gordon (ed.), Pigment cell biology. Academic the enzyme molecule and bind its copper moiPress Inc., New York. ety. DDC is the only compound we have tested 6. Menon, I. A., and H. F. Haberman. 1968. Tyrosinase activity in serum from normal and melanoma bearing so far which completely suppressed the o-dimice. Cancer Res. 28:1237-1241. phenoloxidase of M. leprae. This potency of 7. Pomerantz, S. H. 1966. The tyrosine hydroxylase activity DDC is explained by the presence of the two of mammalian tyrosinase. J. Biol. Chem. 241:161-168. nonpolar ethyl groups in the DDC molecule 8. Prabhakaran, K. 1971. Unusual effects of reducing agents on o-diphenoloxidase of Mycobacterium leprae. J. which more or less shadow the sulfurs which Bacteriol. 107:787-789. constitute the polar region (13). The ethyl 9. Prabhakaran, K. 1973. Dopa metabolism by Mycobactegroups being lipid soluble would enable the rium leprae: its implications in culture of the bacillus compound to pass through lipid predominant and chemotherapy of leprosy. Lepr. Rev. 44:112-119. pores easily. It may be mentioned that similar 10. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. 1969. Effect of inhibitors on phenoloxidase of Mycobacresults were obtained with intact as well as terium leprae. J. Bacteriol. 100:935-938. undisIDDC penetrated disrupted bacteria. 11. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. rupted bacteria, whereas the other compounds 1972. The nature of the phenolase enzyme in Mycobacterium leprae: structure-activity relationships of subdid not. strates and comparison with copper proteins and enThe data presented above are in agreement zymes. Microbios 5:273-281. M. leprae sepa- 12. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. with our earlier studies using rated from lepromatous human tissues (8, 10). 1973. Particulate nature of o-diphenoloxidase in Mycobacterium leprae and assay of the enzyme by the These findings confirm that o-diphenoloxidase radioisotope technique. Microbios 8:151-157. activity is an intrinsic characteristic of the 13. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. leprosy bacillus. The results also provide addi1975. Hypopigmentation of skin lesions in leprosy and tional evidence that the bacteria recovered from occurrence of o-diphenoloxidase in Mycobacterium leprae. In V. Riley (ed.), Proceedings of the IX Interthe infected armadillos are in fact M. leprae. So national Pigment Cell Conference. S. Karger, Basel. far, we have detected oxidation of dopa by 14. Prabhakaran, K., and W. F. Kirchheimer. 1966. Use of bacilli separated from skin nodules, liver, and 3,4-dihydroxyphenylalanine oxidation in the identification of Mycobacterium leprae. J. Bacteriol. spleen of armadillos, footpads of mice, and skin 92:1267-1268. nodules, spleen, and testes of leprosy patients. K., W. i'. Kirchheimer, and E. B. Harris. No other mycobacteria have shown phenoloxi- 15. Prabhakaran, 1968. Oxidation of phenolic compounds by Mycobactebe dase activity (1), and the enzyme could not rium leprae and inhibition of phenolase by substrateanalogues and copper chelators. J. Bacteriol. induced in cultivable mycobacteria by adding a 95:2051-2053. phenolic substrate to the culture medium (10).
AND
Vol. 12, No. 2 Printed in U.S.A.
IMMUNITY, Aug. 1975, p. 267-269
Copyright 0 1975 American Society for Microbiology
o-Diphenoloxidase of Mycobacterium leprae Separated from Infected Armadillo Tissues K. PRABHAKARAN,* E. B. HARRIS,
AND W.
F. KIRCHHEIMER
U.S. Public Health Service Hospital, Carville, Louisiana 70721
We reported earlier the occurrence of a unique o-diphenoloxidase in Mycobacterium leprae recovered from lepromatous human tissues. No other source of M. Ieprae for biochemical studies was available at the time. In the present report, properties of phenoloxidase in M. Ieprae separated from infected armadillo tissues are presented. The results show that the o-diphenoloxidase remains unaltered in the passage of the bacilli from the human to the animal host, indicating that the enzyme is an intrinsic characteristic of the leprosy bacteria.
The only information available in literature on the metabolic properties of Mycobacterium leprae is a series of studies we reported using concentrates of organisms separated from lepromatous human tissues (8-15). No authenticated culture of the bacillus is as yet available, and until recently no immunologically unimpaired animal model was known which, when inoculated with the leprosy bacteria, develops the heavy systemic form of the disease. Now it has been shown that M. Ieprae produces disseminated leprosy in the nine-banded armadillo (Dasypus novemcinctus Linn.) (2). We found that organisms separated from lepromatous human tissues contain an unusual form of the enzyme o-diphenoloxidase (o-diphenol:oxygen oxidoreductase [EC1.10.3.1]). The o-diphenoloxidase occurring in mammalian melanocytes oxidizes L-dopa (3,4-dihydroxyphenyalanine) to dopachrome (2-carboxy-2,3-dihydroindole-5,6quinone). Dopachrome is characterized by an absorbance maximum at 475 nm in the spectrum (5). The mammalian enzyme shows little activity towards D-dopa or derivatives of dopa like epinephrine and norepinephrine. The enzyme in M. leprae converted both D- and Ldopa to indole-5,6-quinone and also oxidized a variety of phenolic substrates to quinones (11). Indole-5,6-quinone is distinguished from dopachrome by its absorbance maximum at 540 nm (5). Since at the time of our earlier studies lepromatous human tissues were the only sources of M. leprae for biochemical studies, it was not possible to ascertain whether phenoloxidase is a constitutive enzyme in the bacillus or an enzyme induced by the milieu in which the organism proliferates. The present report deals with the o-diphenoloxidase of M. leprae separated from infected tissues of armadillos. The results show that the o-diphenoloxidase is a constitutive enzyme in M. leprae, which remains unaltered in the passage of bacilli from the human to the animal host.
MATERIALS AND METHODS
Organisms. Concentrates of M. leprae were separated from infected liver of armadillos. The tissues were collected aseptically and kept frozen at -20 or -80 C. The preparative procedure has been described earlier and is carried out at 0 to 2 C (8, 13). It involves homogenization of the tissue and differential and density gradient centrifugations of the homogenate in inert solutions, such as those of sucrose and KCI. The bacilli were disrupted by ultrasonic oscillation (12). Some preparations of the bacilli were treated with trypsin, acetone, ether, and dilute NaOH to remove any host tissue materials adsorbed superficially. These treated organisms also readily oxidized dopa. Liver tissue by itself did not oxidize either L- or D-dopa. In bacilli recovered from tissues of animals which had been dead for several hours, phenoloxidase was very low and rather inconsistent. Prolonged storage of the tissues at -20 or -80 C also resulted in considerable loss in the activity of the enzyme in the bacteria. Enzymes. Lyophilized mushroom tyrosinase (odiphenoloxidase) was purchased from Sigma Chemical Co. (St. Louis, Mo.) or from the ICN Life Sciences Group (Cleveland, Ohio). Cultured melanocytes served as a source of mammalian o-diphenoloxidase. The melanocyte cultures (derived from the Cloudman S-91 melanoma) were obtained from the American Type Culture Collection. The cells were grown at 37 C in Ham F-10 medium containing 10% fetal calf serum. Chemicals. D-Dopa was purchased from the ICN Life Sciences Group, and L-dopa was obtained from Sigma. DL- [G-3Hjdopa (labeling distributed generally, 250 mCi/mmol) was obtained from the Amersham-Searle Corp. (Arlington Heights, Ill.). Glass counting vials were purchased from Beckman Instruments, Inc. (Fullerton, Calif.), and the scintillation solution (Aquasol) was obtained from New England Nuclear Corp. (Boston, Mass.). Other chemicals used were those of the highest purity commercially available. Enzyme assay. Dopa is converted to quinone and water by o-diphenoloxidase, and oxygen is consumed in the process. When tritium-labeled dopa was used as substrate, activity of the labeled water formed was measured in a liquid scintillation counter. Details of the procedure have been described before (6, 12), 267
INFECT . IMMUN .
PRABHAKARAN, HARRIS, AND KIRCHHEIMER
268
which has been modified from the method originally reported by Pomerantz (7). The reaction system consisted of: Na,HPO4-KH,PO4 buffer (pH 6.8), 40 glmol; DL- [G-3H]dopa, 1 uCi; "cold" (unlabeled) L-dopa, 1 Mmol; M. leprae, 1.2 mg of protein; temperature, 37 C; time, 60 min; volume, 2 ml. Appropriate controls were run of heat-inactivated enzyme (100 C, 30 min) with substrate and of substrate without enzyme. Formation of quinone from dopa was assayed by determining the spectrum of the reaction mixture, as described before (11). The reaction system consisted of the following constituents in the final concentrations indicated: Na,HPO4-KH,P04 buffer (pH 6.8), 0.1 M; substrates (except epinephrine), 0.002 M; epinephrine, 0.0005 M; inhibitors, 0.004 M; M. leprae, 1.5 to 2.0 mg of protein; melanoma cells, 2 mg of protein; mushroom tyrosinase, 10 Ag; temperature, 37 C; time, 30 min; volume, 3 ml. We measured the absorbance maximum for dopachrome at 480 nm; it was identical to the value at 475 nm, since dopachrome gives a broad peak in the area. We have reported previously the uptake of oxygen by M. leprae in the oxidation of dopa (13). The effect of inhibitors on o-diphenoloxidase of M. leprae was assayed spectrophotometrically by comparing the absorbance maxima of the reaction products with and without the inhibitors (8).
RESULTS AND DISCUSSION Oxidation of substrates. The oxidation of substrates by o-diphenoloxidase from different sources is shown in Table 1. Mushroom tyrosin-
well as M. Ieprae recovered from armadillo tissues converted both D- and L-dopa to quinone at the same rate and also oxidized epinephrine and norepinephrine. These data are in agreement with those obtained with bacilli from human tissues (11). Mammalian tyrosinase oxidized L-dopa; however, it was inactive towards D-dopa and the catecholamines. Epinephrine undergoes rapid auto-oxidation at pH 6.8; to avoid this, the reaction was carried out at pH 6.5. Table 2 illustrates the oxidation of tritium-labeled dopa by M. leprae obtained from the armadillo. Heating the bacilli at 100 C for 30 min produced considerable loss of activity, indicating that the enzyme is labile, although some residual activity was still present. The effect of reducing agents on o-
diphenoloxidase from different sources is presented in Table 3. Reduced glutathione, ascorbic acid, and cysteine completely inhibited plant and mammalian tyrosinases. Reducing agents are known to be inhibitors of tyrosinase (3). However, these compounds showed little effect on the phenoloxidase of M. Ieprae. The bacilli used had been disrupted by ultrasonic oscillation. As such, the results observed are not due to the permeability barrier of the bacterial cell membrane. Apparently the active site of the enzyme in M. leprae is not readily accessible to the inhibitors, unlike in plant and mammalian tyrosinases. This phenomenon is further borne out by data on the effect of metal chelators on the enzyme from different sources, which are presented in Table 4. o-Diphenolkidase is known to be a copper protein (4). Cyanide is a potent inhibitor of metallo enzymes. Diethyldithiocarbamate (DDC) and pencillaTABLE 2. Oxidation of tritium-labeled dopa by M. leprae
a
ase as
['H Idopa
Bacilli
Total
oxidized (pmol)
Unheated Heated
132,159a 31,803
238.13 57.31
Values are corrected for auto-oxidation of dopa. TABLE 3. Effect of reducing agents on o-diphenoloxidase from different sources Reaction system
Enzyme + dopa Enzyme + dopa + ascorbate Enzyme + dopa + GSHa Enzyme + dopa + cysteine a
480 nm Absorbance Absorbance ateaoct at 540 nmMuhom (M. leprae) Mushroom Melanocyte tyrosinase
culture
0.108 0.098
0.120 0
0.140 0
0.080
0
0
0.095
0
0
GSH, Reduced glutathione.
* TABLE 1. Oxidation of dopa and its derivatives by M. leprae and by plant and mammalian tyrosinase: increase in absorbancea Substrate
M. leprae
Mushroom tyrosinase
Melanocyte
L-Dopa D-Dopa L-Epinephrine DL-Norepinephrine
0.100 (540)" 0.099 (540) 0.100 (480) 0.060 (480)
0.095 (480) 0.100 (480) 0.240 (480) 0.140 (480)
0.160 (480) 0 0 0
culture
a Absorbance values are corrected for absorbance of enzyme and nonenzymatic oxidation of substrates. h Values in parentheses indicate absorbance maxima in nanometers.
o-DIPHENOLOXIDASE IN ARMADILLO BACTERIA
VOL. 12, 1975
TABLE 4. Effect of metal chelators on o-diphenoloxidase from different sources Reaction system Enzyme + dopa Enzyme + dopa + NaCN Enzyme + dopa + penicillamine Enzyme + dopa + DDC
Absorbance Absorbance at 480 nm at 540 nm Mushroom Melanocyte (M. leprae) tyrosinase culture
0.099 0.095
0.130 0
0.140 0
0.100
0
0
0
0
0
mine are effective copper chelators, used in the treatment of hepatolenticular degeneration in man. All the three metal chelators produced total inhibition of mammalian and plant tyro-
269
The exact metabolic role of this unusual enzyme in the survival and proliferation of the leprosy organism still remains to be elucidated. ACKNOWLEDGMENTS This investigation was supported by grants from the U.S.-Japan Cooperative Medical Science Program of the National Institute of Allergy and Infectious Diseases (AI-07890 and AI-11204 BM) and by the World Health Organization.
LITERATURE CITD 1. Kirchheimer, W. F., and K. Prabhakaran. 1968. Metabolic and biologic tests on mycobacteria once labeled as leprosy bacilli. Int. J. Lepr. 36:162-165. 2. Kirchheimer, W. F., and E. E. Storrs. 1971. Attempts to establish the armadillo (Dasypus novemcinctus Linn.) as a model for the study of leprosy. I. Report of lepromatoid leprosy in an experimentally infected armadillo. Int. J. Lepr. 39:693-702. 3. Lemer, A. B., and T. B. Fitzpatrick. 1950. Bichemistry of melanin formation. Physiol. Rev. 30:91-126. 4. Makino, N., P. McMahill, and H. S. Mason. 1974. The oxidation state of copper in resting tyrosinase. J. Biol.
sinases. However, penicillamine and cyanide showed little effect on the o-diphenoloxidase activity of the bacilli. Probably, the configuraChenh 249:6062-6066. tion of the enzyme protein in M. leprae is such Structure of melanins, p. 563-582: that these inhibitors are unable to penetrate 5. Mason, H. S. 1959. In M. Gordon (ed.), Pigment cell biology. Academic the enzyme molecule and bind its copper moiPress Inc., New York. ety. DDC is the only compound we have tested 6. Menon, I. A., and H. F. Haberman. 1968. Tyrosinase activity in serum from normal and melanoma bearing so far which completely suppressed the o-dimice. Cancer Res. 28:1237-1241. phenoloxidase of M. leprae. This potency of 7. Pomerantz, S. H. 1966. The tyrosine hydroxylase activity DDC is explained by the presence of the two of mammalian tyrosinase. J. Biol. Chem. 241:161-168. nonpolar ethyl groups in the DDC molecule 8. Prabhakaran, K. 1971. Unusual effects of reducing agents on o-diphenoloxidase of Mycobacterium leprae. J. which more or less shadow the sulfurs which Bacteriol. 107:787-789. constitute the polar region (13). The ethyl 9. Prabhakaran, K. 1973. Dopa metabolism by Mycobactegroups being lipid soluble would enable the rium leprae: its implications in culture of the bacillus compound to pass through lipid predominant and chemotherapy of leprosy. Lepr. Rev. 44:112-119. pores easily. It may be mentioned that similar 10. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. 1969. Effect of inhibitors on phenoloxidase of Mycobacresults were obtained with intact as well as terium leprae. J. Bacteriol. 100:935-938. undisIDDC penetrated disrupted bacteria. 11. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. rupted bacteria, whereas the other compounds 1972. The nature of the phenolase enzyme in Mycobacterium leprae: structure-activity relationships of subdid not. strates and comparison with copper proteins and enThe data presented above are in agreement zymes. Microbios 5:273-281. M. leprae sepa- 12. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. with our earlier studies using rated from lepromatous human tissues (8, 10). 1973. Particulate nature of o-diphenoloxidase in Mycobacterium leprae and assay of the enzyme by the These findings confirm that o-diphenoloxidase radioisotope technique. Microbios 8:151-157. activity is an intrinsic characteristic of the 13. Prabhakaran, K., E. B. Harris, and W. F. Kirchheimer. leprosy bacillus. The results also provide addi1975. Hypopigmentation of skin lesions in leprosy and tional evidence that the bacteria recovered from occurrence of o-diphenoloxidase in Mycobacterium leprae. In V. Riley (ed.), Proceedings of the IX Interthe infected armadillos are in fact M. leprae. So national Pigment Cell Conference. S. Karger, Basel. far, we have detected oxidation of dopa by 14. Prabhakaran, K., and W. F. Kirchheimer. 1966. Use of bacilli separated from skin nodules, liver, and 3,4-dihydroxyphenylalanine oxidation in the identification of Mycobacterium leprae. J. Bacteriol. spleen of armadillos, footpads of mice, and skin 92:1267-1268. nodules, spleen, and testes of leprosy patients. K., W. i'. Kirchheimer, and E. B. Harris. No other mycobacteria have shown phenoloxi- 15. Prabhakaran, 1968. Oxidation of phenolic compounds by Mycobactebe dase activity (1), and the enzyme could not rium leprae and inhibition of phenolase by substrateanalogues and copper chelators. J. Bacteriol. induced in cultivable mycobacteria by adding a 95:2051-2053. phenolic substrate to the culture medium (10).
