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Trimethylamine oxidase of nurse shark liver and its relation to mammalian mixed function amine oxidase
Comp. Biochem. Physiol., 1973, VoL 45B, pp. 895 to 903. Pergamon Press. Printed in Great Britain
TRIMETHYLAMINE OXIDASE OF NURSE SHARK LIVER AND ITS RELATION TO MAMMALIAN MIXED FUNCTION AMINE OXIDASE* L E O N G O L D S T E I N and SUSAN D E W I T T - H A R L E Y Division of Biomedical Science, Brown University, Providence, R.I. 02912 and the Lerner Marine Laboratory, Biminl, The Bahamas
(Received 4 December 1972) Abatract--1. Trimethylamine (TMA) oxidase activity was characterized in
nurse shark (C,inglymostoma cirratum) liver homogenates. Enzyme activity was absolutely dependent on NADPH and was localized mainly in the microsomes. 2. Inhibitors of cytochrome oxidase (azide) and cytochrome P-450 (CO and SKF-525A) had little or no effect on TMA oxidase activity. 3. The tertiary amines, N,N-dimethylaniline and chlorpromazine, inhibited TMA oxidation. 4. N,N-dimethylaniline was oxidized readily by liver homogenates from nurse shark and lemon shark but only slightly by homogenates from spiny dogfish and little skate. A similar pattern was found for the oxidation of trimethylamine. INTRODUCTION TPaMgrI-rCL~VnNE OXIDE (TMAO) occurs in relatively high concentrations ( ~ 7 0 raM) in the body fluids of elasmobranchs (Groninger, 1959) where it is thought to play an osmoregulatory role. The origin of this compound in elasmobranchs is not clear, but we have recently shown (Goldstein & Funkhouser, 1972) that it can be synthesized endogenously in the nurse shark, Ginglymostoma cirraturn, via a pathway involving the enzyme trimethylamine (TMA) oxidase. On the basis of some preliminary experiments done on liver homogenates we suggested that T M A oxidase of elasmobranch liver may be similar to the mammalian enzyme system involved in the N-oxidation of tertiary amines. The latter enzyme system, which is capable of catalyzing the oxidation of a variety of foreign tertiary amines including several drugs, has been purified and characterized in detail (Ziegler & Mitchell, 1972). However, as with most drug metabolizing enzymes, the evolutionary origin and biological function of this system is not clear. It is conceivable that this system was present in primitive vertebrates, where it functioned in the synthesis of T M A O from T M A and this same system may have been retained during vertebrate evolution and been put to a new use--the detoxification of foreign tertiary amines, when the vertebrates entered new environments and encountered * Supported by N.S.F. Grant No. GB30357X. 895
896
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
foreign nitrogenous compounds. I f one could show that basic similarities exist between the properties of mammalian mixed function amine oxidase and T M A oxidase of elasmobranchs, this theory would gain support. I n the present study we examined the properties of T M A oxidase in homogenates of nurse shark liver and compared these properties with those reported for the mammalian mixed function amine oxidase. These studies show that the two enzyme systems share m a n y c o m m o n properties and support the theory that the mammalian mixed function amine oxidase evolved from the enzyme system involved in the biosynthesis of T M A O in lower vertebrates. MATERIALS AND METHODS Nurse sharks (Ginglymostoma cirratum), lemon sharks (Negaprion brevirostris) and stingrays (Dasyatis americana) weighing 2--4 kg, were captured in the vicinity of Bimini, Bahamas. Skates (Raja erinacea) 0"5-1"0 kg, and the dogfish (Squalus acanthias), 1-3 kg, were captured off Mount Desert Island, Maine. The fish were sacrificed within 1 week of capture, the livers removed and either assayed immediately, or frozen on dry ice and maintained below - 70°C until use. Livers of nurse and lemon sharks could be stored for weeks at this temperature with little loss of TMA oxidase activity. For the assay of TMA oxidase, livers were homogenized in 9 vol. of ice-cold, 0"1 M potassium pyrophosphate (pH 7"8) and enzyme assays performed in the standard incubation medium (3"0ml) containing 120/xmoles potassium pyrophosphate, pH 7.8; NADPH (Sigma), 1.5 mg; glucose-6-phosphate (Sigma), 6 nag; glucose-6-phosphate dehydrogenase (Sigma), 0.6 units; trimethylamineJ4C'HC1 (N.E. Nuclear), sp. act. 1"0/~Ci//zM), 0"15 ~Ci; trimethylamine'HCl (Sigma), pH 7"8, 3/zmoles; MgC12, 30/zmoles; and 30-60 mg tissue. The reaction was started by the addition of tissue homogenate and carried out in an atmosphere of 100% O 2 at 37-38°C. The standard assay conditions were modified, as described in the Tables and Figures, by the addition of inhibitors and by varying the pH and trimethylamine.HC1 concentrations. The reaction was stopped by the addition of 0"1 vol. of 50% trichloracetic acid, the mixture allowed to stand on ice for 10 min and then centrifuged. The supernatant fluid was extracted with hydrated ether to remove the trichloracetic acid and analyzed for trimethylamine-t~C-oxide as described by Goldstein & Funkhouser (1972). Mixed fimction amine oxidase was assayed in liver homogenates by a modification of the procedure of Ziegler & Pettit (1964). The incubation mixture contained 250/~moles HEPES (n-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid) buffer, pH 7"6; 12"5/~moles MgC12; 1"75 pmoles NADP; 12"5 pmoles glucose-6-phosphate; 2 units glucose-6-phosphate dehydrogenase; 117 pmoles semicarbazide; 20/~moles N,N-dimethylaniline (MCB); and 160 mg tissue in a volume of 2"5 ml. The mixture was incubated for 30 or 60 min at 37-38°C in an atmosphere of 100% 02. The reaction was stopped by the addition of 1-0 ml ZnSO4 (8"9%), 1"5 ml of a saturated solution of Ba(OH)2 and 0"5 ml of saturated Na2B40~ for 30 rain and analyzed for N,N-dimethylaniline oxide as follows: 6"0 ml of the resulting supernatant fluid was placed in a 45 ml extraction tube (Kontes Glass Co.) and the pH adjusted to about 9"4 by the addition of 0"2"ml 5 M NH4OH. The alkaline solution was extracted three times with 10 ml of peroxide free, hydrated ether to remove N,N-dimethylaniline. Four ml of the aqueous layer was adjusted to approximately pH 2"5 with a few drops of 3 M trichloroacetic acid and 1"0 ml of 0-68 M NaNO2 was added. The solution was heated for 10 rain at 60°C, mixed well and cooled to room temperature. The optical density of the solution was determined at 420 nm in a Spectronic 20 spectrophotometer and the resulting O.D. converted to ftmoles with the aid of a standard curve prepared with N,N-dimethylaniline. The values were corrected for endogenous chromogens and non-specific oxide
NtmSE SHARKTMA OXIDASE
897
formation, using control flasks treated in a manner identical to the experimental flasks except that the enzyme reaction was stopped immediately after the flasks had been placed in the incubation bath. RESULTS
Pyridine nucleotide dependence of TMA oxidase Conversion of 14C-TMA to 14C-TMAO by liver homogenates was absolutely dependent on the presence of N A D P H , which was continually generated by the addition of glucose-6-phosphate (G-6-P), G-6-P dehydrogenase and N A D P to the incubation medium (Table 1). A N A D H generating system was only 6 per cent as effective as the N A D P H system in supporting this reaction. TABLE 1--PYRIDINE NUCLEOTIDE REQUIREMENT OF TRIMETHYLAMINE OXIDASE
Pyridine nucleotide added None NADPH* NADHt
TMA-14C-O production (dis/min per g liver x hr)
Activation + nucleotide - nucleotide
2-17 x 104 1"22 x 10e 5.51 x 104
-56"0 2"5
Values are means of duplicate assays. *Added as NADPH generating system; NADP, 0"65 mM; glucose-6-phosphate, 7.09 mM; glucose-6-phosphate dehydrogenase, 0.2 units/m1. tAdded as NADH generating system: NAD, 3"02 mM; fl-hydroxybutyrate, 3-33 mM; fl-hydroxybutyrate dehydrogenase, 0"4 units/m1.
Subcellular distribution of TMA oxidase Differential centrifugationstudies done on homogenatesof frozen livers showed that most of the T M A oxidase activity in nurse shark liver was localized in the microsomal fraction (Table 2). Approximately a quarter of the activity was found in the cytosol fraction. T h e latter figure is probably an overestimate of the percentage total activity in the cytosol fraction. Freezing and thawing of the liver TABLE 2 - - L o c A L I Z A T I O N OF TRIMETHYLAMINE OXIDASE IN LIVER HOMOGENATES
Fraction
Total counts in fraction
Total activity (%)
Homogenate Nuclei Mitochondria Microsomes Cytosol
6"89 x 105 2-32 x 104 3-73 x 108 3"66 x 105 1 "54 x 105
-4 1 67 28
Assays done on livers previously stored at -85°C.
898
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
undoubtedly caused some disruption of microsomal membranes and loss of these membranes from the microsomal to the cytosol fraction. T h e nuclear and mitochondrial fractions contained negligible enzyme activity.
Effects of cytochrome inhibitors on T M A oxidase activity Cytochrome inhibitors had little effect on the formation of T M A O from T M A (qQable 3). Sodium azide, a cytochrome oxidase inhibitor, had a slight inhibitory effect on T M A O formation. Similarly SKF-525A, a competitive inhibitor of TABLE 3 - - L A C K
OF EFFECT OF CYTOCHROME INHIBITORS ON TRIMETHYLAMINE OXIDASE
Experiment Assay medium TMA-14C-O production Inhibition No. Inhibitor concentration (dis/rain per g liver x hr) (°'o) 1
2 3*
None Sodium Azide
2 x 10 -3 M
3"61 × l0 s
11
None SKF-525At
2 x 10 -3 M
1"03 × 106 1"07 × 10°
0
CO-O~ 2 : 1 4 :1 10 : 1
2"37 x 2"05 x 2"56 x 1"95 x
None CO
4"07 x 105
105 105 105 105
14 0 18 Average
11
Values are means of duplicate assays. *Assays done on livers previously stored at -85°C. t2-Diethylaminoethyl 2,2-diphenylvalerate HC1. cytochrome P-450, had a slight inhibitory effect. T h e inhibitions produced by both agents were marginal and of questionable significance, considering the crude state of the enzyme system in the tissue homogenate. Carbon monoxide, another inhibitor of cytochrome P-450, was tested in CO/O2 ratios ranging from 2 : 1 to 10 : 1. T h e average inhibition produced at the three ratios tested was 11 per cent. T h e significance of this inhibition is questionable, not only because of its magnitude but also due to the lack of correlation between the CO/O 2 ratio and the percentage inhibition of enzyme activity (Table 3).
Effect of foreign tertiary amines on TIYIA oxidase T w o foreign tertiary amines, N,N-dimethylaniline (DMA) and chlorpromazine, were tested as inhibitors of shark liver T M A oxidase activity. As shown in Table 4, both compounds produced significant inhibitions of T M A O formation in liver homogenates, D M A being the more potent of the two inhibitors.
mrRsE SH~aK T M A OXlD~
899
TABLE 4---EFFECT OF N,N-DIMErHYLANILINE (DMA) AND CHLORPROMAZINE ON TRIMETHYLAMINE OXIDASE
Experiment Assay medium TMA-X4C-O production Inhibition No. Inhibitor concentration (dis/min per g liver x hr) (%) 1
None DMA
2
None Craorpromazine*
1'03 x 10a 1"87 x 104
5 x 10 -3 M
98
2"51 x 105 1"44 x l0 s
2x10-3M
43
Values are means of duplicate assays. *2-Chloro-10-(3-dimethyl-aminopropyl)phenothiazine.
N,N-dimethylaniline oxidation by elasmobranch liver homogenates The ability of liver homogenates of several elasmobranchs to oxidize N,Ndimethylaniline (DMA) is shown in Table 5. DMA was readily oxidized by liver homogenates from lemon sharks and nurse sharks but to only a slight degree by liver homogenates from dogfish and skate. As shown in the table this pattern of DMA metabolism corresponds exactly to that found for the oxidation of TMA to TMAO by liver homogenates from these same species. TABLE 5--MIx~
FUNCTION (MFA) AMINE OXIDASE AND TRIMETHYLAMINE ACTIVITIES IN LIVERS OF ELASMOBRANCH FISHES
Species
Nurse shark Lemon shark Spiny dogfish Little skate
MFA oxidase
(TMA)
OXIDASE
TMA oxidase
/zmoles/g liver x hr 1-04 (2) 6"3 (2) 1.41 (4) 8.1 (4) 0"108 (2) 0"0042 (2) 0"051 (2) 0"0051 (2)
Values are averages of two to four assays (number shown in parentheses) on livers from one or two fish each.
pH optimum and substrate affinity The pH optimum and TMA Km were determined for TMA oxidase of nurse shark liver. As shown in Fig. 1, the pH optimum was approximately 9-0 in both Tris and glycine buffers. The TMA Km determined at pH 9-0 was 1.5 x 10 -4 M. DISCUSSION
Although the occurrence of trimethylamine oxide (TMAO) in vertebrate tissues has been known for many years, its biosynthesis was only fairly recently
900
LEON GOLDSTEINAND SUSAN DEWITT-HARLEY
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FIo. 1. pH activity curves of TMA oxidase of nurse shark liver. p H ' s were measured at incubation temperature--37-38°C. demonstrated (Baker & Chaykin, 1962). They showed that hog liver microsomes catalyzed the oxidation of T M A (trimethylamine) to TMAO. The enzyme was found in the livers of a number of vertebrates (Baker et al., 1963), although its occurrence in some groups, e.g. fishes, was reported to be sporadic. Thus the enzyme probably plays an important role, such as T M A detoxification, in vertebrates. The properties of the hog liver T M A oxidase reported by Baker & Chaykin are similar to those of mixed function amine oxidase (MFA), an enzyme system found in mammalian liver that catalyzes the oxidation of a number of tertiary amines to their respective N-oxides (Ziegler et al., 1969). As both enzyme systems are localized in the microsomes, require NADPH and molecular O3 for activity and are able to oxidize T M A to TMAO, it is quite possible that mammalian T M A oxidase and MFA oxidase are really the same enzyme system. Our previous studies (Goldstein & Funkhouser, 1972) on T M A oxidase in the liver of the nurse shark suggested that the enzyme in elasmobranch liver may also share common properties with mammalian MFA oxidase. If this were true then certain evolutionary implications could be derived. Therefore, we systematically determined the characteristics of T M A oxidase in nurse shark liver homogenates and compared these with the properties of mammalian MFA oxidase. As is the case with mammalian MFA oxidase, nurse shark oxidase is localized in the microsomal fraction of the liver, the enzyme system requires NADPH for activity and this requirement cannot be replaced by NADH. Neither enzyme is inhibited by inhibitors of cytochrome oxidase (azide) or P-450 (CO and SKF-525A). The pH optimum of the shark enzyme system is approximately 9.0 while that of hog liver is about 8.5. The T M A Km for the shark enzyme is approximately 0.1 raM. A T M A K m has not been reported for the hog liver MFA oxidase. However, the studies of Baker & Chaykin (1962) on T M A oxidation by hog liver microsomes indicate that the K m of the mammalian enzyme system is the same order as that
NURSE SHARK T M A
OXtD~E
901
found in the nurse shark. Thus, nurse shark T M A oxidase and hog liver M F A oxidase appear to be quite similar in all parameters measured. Further evidence for similarity of the two enzymes is the distribution of the two enzyme activities in the livers of different elasmobranchs. Livers of the two elasmobranchs that were able to oxidize T M A to T M A O were also capable of converting N,N-dimethylaniline (DMA) (a substrate of mammalian M F A oxidase) to DMAO while livers of the other two elasmobranchs examined (dogfish and skate) had only limited capability of oxidizing either substrate. The evolutionary origin and biological function of the mammalian drug metabolizing enzymes are not clear. Some of these drug metabolizing enzymes have been reported to be present in primitive classes of vertebrates such as the elasmobranchs (Adamson & Guarino, 1972), which are thought to have changed little since their first appearance approximately 400 million years ago. It is highly unlikely that the drug metabolizing enzymes evolved to detoxify the wide variety of foreign compounds that are capable of being metabolized by these enzyme systems. It is much more plausible that these enzymes evolved for different functions and coincidentally possessed the ability to detoxify foreign compounds. For example, the steroid hydroxylating enzymes which are found in both lower and higher vertebrates share a number of properties in common with the drug metabolizing enzymes--microsomal localization, NADPH requirement, CO inhibition and induction by methylcholanthrene and phenobarbital (Conney et al., 1969). Thus, it is quite likely that these enzymes evolved for the purpose of hydroxylating endogenous steroids and possessed low enough specificity to enable them to accept a number of foreign compounds as substrates. Similarly, in the present study we have shown that the enzyme system catalyzing the oxidation of T M A to T M A O in shark liver shares many properties in common with the mammalian mixed function amine oxidase. We propose that the original function of this enzyme system was the N-oxidation of T M A (and possibly other biological amines), and that the ability of the system to oxidize foreign amines is due either to a low specificity of T M A oxidase or to a secondary adaptation of the enzyme system. A more detailed comparison of the two enzymes and purification of the shark enzyme is needed before these two alternatives can be distinguished. The sporadic distribution of the T M A oxidase in elasmobranchs is puzzling. As shown in Table 6, the enzyme is found in the nurse shark, lemon shark and smooth dogfish but is absent in the spiny dogfish, little and big skates and the American stingray. There is not a clear pattern to its distribution; its occurrence does not appear to be restricted to any group of elasmobranchs or any geographical location. We had originally suggested that the enzyme might occur in elasmobranchs inhabiting tropical waters (Goldstein & Funkhouser, 1972) but its absence in the American stingray, which is found in both temperate and tropical water, weakens this theory. However, it may be noted that among the six elasmobranchs studied, the three possessing this enzyme are found in temperate and tropical waters. At any rate it is highly unusual to find such sporadic distribution of an enzyme within a single group of animals, although Adamson & Guarino (1972)
902
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
TABLE 6--CONVERSION OF TRIMETHYLAMINE TO TRIMETHYLAMINE OXIDE IN ELASMOBRANCHS
Species Lemon shark Nurse shark Smooth dogfish American stingray Spiny dogfish Little skate
In vivo*
In vitrot
+ + + -
+ + N.T. N.T. + +
*Assayed by conversion of injected a4C-trimethylamine to a4C-trimethylamine oxide. tAssayed by conversion of 14C-trimethylamine to 14C-trimethylamine oxide in fortified liver homogenates. + , Activity present; - , activity absent; +, questionable activity; N.T., not tested.
reported similar findings for the distribution of the drug metabolizing enzyme nitroreductase in elasmobranchs. T h e enzyme was absent in the lemon shark, spiny dogfish and American stingray, but was present in the little skate. It would be interesting to determine the nature of the underlying factors controlling the distribution of T M A oxidase and other drug metabolizing enzymes in this important group of vertebrates. Acknowledgement--The authors thank Miss Anne Backus for skilful technical assistance in several phases of this study; Miss T. Devereux of the National Institute of Environmental Health Sciences for making available her procedure for assay of mixed function amine oxidase; and Dr. Ralph Miech for the sample of SKF-525A.
REFERENCES
ADAMSON R. H. & GUARINOA. M. (1972) The effect of foreign compounds on elasmobranchs and the effect of elasmobranchs on foreign compounds. Comp. Biochem. Physiol. 42A, 171-182. BAKER J. R. & CHAYKINS. (1962) The biosynthesis of trimethylamine-N-oxide. J. biol. Chem. 237, 1309-1313. BAKERJ. R., STRImMPLERA. & CHAYKINS. (1963) A comparative study of trimethylamineN-oxide biosynthesis. Biochim. biophys. Acta 71, 58-64. CONNEYA. H., LEVlN W., JACOBSONM. & KONTZMANR. (1969) Specificity in the regulation of the 6fl, 70~ and 16~-hydroxylation of testosterone by rat liver microsomes. In Symposium on Microsomes andDrug Oxidation (Edited by GILLETTEJ. R., CONNEYA. H., COSMIDES G. J., ESTABROOKR. W . , FOUTS J. R. & MANNERINGG. J.), pp. 279-295. Academic Press, New York. GOLDSTEINL. ~ FUNKHOUSERD. (1972) Biosynthesis of trimethylamine oxide in the nurse shark, Ginglymostoma cirratum. Comp. Biochem. Physiol. 42A, 51-57. GRONINGERH. S. (1959) The occurrence and significance of trimethylamine oxide in marine animals. United States Fish and Wildlife Service, Special Scientific Report. Fisheries No. 333, 1-22.
NURSE SHARK
T M A OXID~E
903
ZIEGLERD. M. & MITCHELL C. H. (1972) Microsomal oxidase--IV. Properties of a mixedfunction amine oxidase isolated from pig liver microsomes. Archs Biochem. Biophys. 150, 116-125. ZIEGLER D. M., MITCHELL C. H. & JOLLOW D. (1969) The properties of purified hepatic microsomal mixed function amine oxidase. In Symposium on Microsomes and Drug Oxidation (Edited by GmLETTE J. R., Co~q'~Y A. H., COSMIDESG. J . , ESTABROOKR. W., FOUTS J. R. & M~NERING G. J.), pp. 173-188. Academic Press, New York. ZXEG~R D. M. & PETTIT F. H. (1964) Formation of an intermediate N-oxide in the oxidative demethylation of N,N-dimethylaniline catalyzed by liver and microsomes. Biochem. biophys. Res. Commun. 15, 188-193.
Key Word Index---Trimethylamine oxidase; amine oxidase; shark liver; Ginglymostoma cirratum.
TRIMETHYLAMINE OXIDASE OF NURSE SHARK LIVER AND ITS RELATION TO MAMMALIAN MIXED FUNCTION AMINE OXIDASE* L E O N G O L D S T E I N and SUSAN D E W I T T - H A R L E Y Division of Biomedical Science, Brown University, Providence, R.I. 02912 and the Lerner Marine Laboratory, Biminl, The Bahamas
(Received 4 December 1972) Abatract--1. Trimethylamine (TMA) oxidase activity was characterized in
nurse shark (C,inglymostoma cirratum) liver homogenates. Enzyme activity was absolutely dependent on NADPH and was localized mainly in the microsomes. 2. Inhibitors of cytochrome oxidase (azide) and cytochrome P-450 (CO and SKF-525A) had little or no effect on TMA oxidase activity. 3. The tertiary amines, N,N-dimethylaniline and chlorpromazine, inhibited TMA oxidation. 4. N,N-dimethylaniline was oxidized readily by liver homogenates from nurse shark and lemon shark but only slightly by homogenates from spiny dogfish and little skate. A similar pattern was found for the oxidation of trimethylamine. INTRODUCTION TPaMgrI-rCL~VnNE OXIDE (TMAO) occurs in relatively high concentrations ( ~ 7 0 raM) in the body fluids of elasmobranchs (Groninger, 1959) where it is thought to play an osmoregulatory role. The origin of this compound in elasmobranchs is not clear, but we have recently shown (Goldstein & Funkhouser, 1972) that it can be synthesized endogenously in the nurse shark, Ginglymostoma cirraturn, via a pathway involving the enzyme trimethylamine (TMA) oxidase. On the basis of some preliminary experiments done on liver homogenates we suggested that T M A oxidase of elasmobranch liver may be similar to the mammalian enzyme system involved in the N-oxidation of tertiary amines. The latter enzyme system, which is capable of catalyzing the oxidation of a variety of foreign tertiary amines including several drugs, has been purified and characterized in detail (Ziegler & Mitchell, 1972). However, as with most drug metabolizing enzymes, the evolutionary origin and biological function of this system is not clear. It is conceivable that this system was present in primitive vertebrates, where it functioned in the synthesis of T M A O from T M A and this same system may have been retained during vertebrate evolution and been put to a new use--the detoxification of foreign tertiary amines, when the vertebrates entered new environments and encountered * Supported by N.S.F. Grant No. GB30357X. 895
896
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
foreign nitrogenous compounds. I f one could show that basic similarities exist between the properties of mammalian mixed function amine oxidase and T M A oxidase of elasmobranchs, this theory would gain support. I n the present study we examined the properties of T M A oxidase in homogenates of nurse shark liver and compared these properties with those reported for the mammalian mixed function amine oxidase. These studies show that the two enzyme systems share m a n y c o m m o n properties and support the theory that the mammalian mixed function amine oxidase evolved from the enzyme system involved in the biosynthesis of T M A O in lower vertebrates. MATERIALS AND METHODS Nurse sharks (Ginglymostoma cirratum), lemon sharks (Negaprion brevirostris) and stingrays (Dasyatis americana) weighing 2--4 kg, were captured in the vicinity of Bimini, Bahamas. Skates (Raja erinacea) 0"5-1"0 kg, and the dogfish (Squalus acanthias), 1-3 kg, were captured off Mount Desert Island, Maine. The fish were sacrificed within 1 week of capture, the livers removed and either assayed immediately, or frozen on dry ice and maintained below - 70°C until use. Livers of nurse and lemon sharks could be stored for weeks at this temperature with little loss of TMA oxidase activity. For the assay of TMA oxidase, livers were homogenized in 9 vol. of ice-cold, 0"1 M potassium pyrophosphate (pH 7"8) and enzyme assays performed in the standard incubation medium (3"0ml) containing 120/xmoles potassium pyrophosphate, pH 7.8; NADPH (Sigma), 1.5 mg; glucose-6-phosphate (Sigma), 6 nag; glucose-6-phosphate dehydrogenase (Sigma), 0.6 units; trimethylamineJ4C'HC1 (N.E. Nuclear), sp. act. 1"0/~Ci//zM), 0"15 ~Ci; trimethylamine'HCl (Sigma), pH 7"8, 3/zmoles; MgC12, 30/zmoles; and 30-60 mg tissue. The reaction was started by the addition of tissue homogenate and carried out in an atmosphere of 100% O 2 at 37-38°C. The standard assay conditions were modified, as described in the Tables and Figures, by the addition of inhibitors and by varying the pH and trimethylamine.HC1 concentrations. The reaction was stopped by the addition of 0"1 vol. of 50% trichloracetic acid, the mixture allowed to stand on ice for 10 min and then centrifuged. The supernatant fluid was extracted with hydrated ether to remove the trichloracetic acid and analyzed for trimethylamine-t~C-oxide as described by Goldstein & Funkhouser (1972). Mixed fimction amine oxidase was assayed in liver homogenates by a modification of the procedure of Ziegler & Pettit (1964). The incubation mixture contained 250/~moles HEPES (n-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid) buffer, pH 7"6; 12"5/~moles MgC12; 1"75 pmoles NADP; 12"5 pmoles glucose-6-phosphate; 2 units glucose-6-phosphate dehydrogenase; 117 pmoles semicarbazide; 20/~moles N,N-dimethylaniline (MCB); and 160 mg tissue in a volume of 2"5 ml. The mixture was incubated for 30 or 60 min at 37-38°C in an atmosphere of 100% 02. The reaction was stopped by the addition of 1-0 ml ZnSO4 (8"9%), 1"5 ml of a saturated solution of Ba(OH)2 and 0"5 ml of saturated Na2B40~ for 30 rain and analyzed for N,N-dimethylaniline oxide as follows: 6"0 ml of the resulting supernatant fluid was placed in a 45 ml extraction tube (Kontes Glass Co.) and the pH adjusted to about 9"4 by the addition of 0"2"ml 5 M NH4OH. The alkaline solution was extracted three times with 10 ml of peroxide free, hydrated ether to remove N,N-dimethylaniline. Four ml of the aqueous layer was adjusted to approximately pH 2"5 with a few drops of 3 M trichloroacetic acid and 1"0 ml of 0-68 M NaNO2 was added. The solution was heated for 10 rain at 60°C, mixed well and cooled to room temperature. The optical density of the solution was determined at 420 nm in a Spectronic 20 spectrophotometer and the resulting O.D. converted to ftmoles with the aid of a standard curve prepared with N,N-dimethylaniline. The values were corrected for endogenous chromogens and non-specific oxide
NtmSE SHARKTMA OXIDASE
897
formation, using control flasks treated in a manner identical to the experimental flasks except that the enzyme reaction was stopped immediately after the flasks had been placed in the incubation bath. RESULTS
Pyridine nucleotide dependence of TMA oxidase Conversion of 14C-TMA to 14C-TMAO by liver homogenates was absolutely dependent on the presence of N A D P H , which was continually generated by the addition of glucose-6-phosphate (G-6-P), G-6-P dehydrogenase and N A D P to the incubation medium (Table 1). A N A D H generating system was only 6 per cent as effective as the N A D P H system in supporting this reaction. TABLE 1--PYRIDINE NUCLEOTIDE REQUIREMENT OF TRIMETHYLAMINE OXIDASE
Pyridine nucleotide added None NADPH* NADHt
TMA-14C-O production (dis/min per g liver x hr)
Activation + nucleotide - nucleotide
2-17 x 104 1"22 x 10e 5.51 x 104
-56"0 2"5
Values are means of duplicate assays. *Added as NADPH generating system; NADP, 0"65 mM; glucose-6-phosphate, 7.09 mM; glucose-6-phosphate dehydrogenase, 0.2 units/m1. tAdded as NADH generating system: NAD, 3"02 mM; fl-hydroxybutyrate, 3-33 mM; fl-hydroxybutyrate dehydrogenase, 0"4 units/m1.
Subcellular distribution of TMA oxidase Differential centrifugationstudies done on homogenatesof frozen livers showed that most of the T M A oxidase activity in nurse shark liver was localized in the microsomal fraction (Table 2). Approximately a quarter of the activity was found in the cytosol fraction. T h e latter figure is probably an overestimate of the percentage total activity in the cytosol fraction. Freezing and thawing of the liver TABLE 2 - - L o c A L I Z A T I O N OF TRIMETHYLAMINE OXIDASE IN LIVER HOMOGENATES
Fraction
Total counts in fraction
Total activity (%)
Homogenate Nuclei Mitochondria Microsomes Cytosol
6"89 x 105 2-32 x 104 3-73 x 108 3"66 x 105 1 "54 x 105
-4 1 67 28
Assays done on livers previously stored at -85°C.
898
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
undoubtedly caused some disruption of microsomal membranes and loss of these membranes from the microsomal to the cytosol fraction. T h e nuclear and mitochondrial fractions contained negligible enzyme activity.
Effects of cytochrome inhibitors on T M A oxidase activity Cytochrome inhibitors had little effect on the formation of T M A O from T M A (qQable 3). Sodium azide, a cytochrome oxidase inhibitor, had a slight inhibitory effect on T M A O formation. Similarly SKF-525A, a competitive inhibitor of TABLE 3 - - L A C K
OF EFFECT OF CYTOCHROME INHIBITORS ON TRIMETHYLAMINE OXIDASE
Experiment Assay medium TMA-14C-O production Inhibition No. Inhibitor concentration (dis/rain per g liver x hr) (°'o) 1
2 3*
None Sodium Azide
2 x 10 -3 M
3"61 × l0 s
11
None SKF-525At
2 x 10 -3 M
1"03 × 106 1"07 × 10°
0
CO-O~ 2 : 1 4 :1 10 : 1
2"37 x 2"05 x 2"56 x 1"95 x
None CO
4"07 x 105
105 105 105 105
14 0 18 Average
11
Values are means of duplicate assays. *Assays done on livers previously stored at -85°C. t2-Diethylaminoethyl 2,2-diphenylvalerate HC1. cytochrome P-450, had a slight inhibitory effect. T h e inhibitions produced by both agents were marginal and of questionable significance, considering the crude state of the enzyme system in the tissue homogenate. Carbon monoxide, another inhibitor of cytochrome P-450, was tested in CO/O2 ratios ranging from 2 : 1 to 10 : 1. T h e average inhibition produced at the three ratios tested was 11 per cent. T h e significance of this inhibition is questionable, not only because of its magnitude but also due to the lack of correlation between the CO/O 2 ratio and the percentage inhibition of enzyme activity (Table 3).
Effect of foreign tertiary amines on TIYIA oxidase T w o foreign tertiary amines, N,N-dimethylaniline (DMA) and chlorpromazine, were tested as inhibitors of shark liver T M A oxidase activity. As shown in Table 4, both compounds produced significant inhibitions of T M A O formation in liver homogenates, D M A being the more potent of the two inhibitors.
mrRsE SH~aK T M A OXlD~
899
TABLE 4---EFFECT OF N,N-DIMErHYLANILINE (DMA) AND CHLORPROMAZINE ON TRIMETHYLAMINE OXIDASE
Experiment Assay medium TMA-X4C-O production Inhibition No. Inhibitor concentration (dis/min per g liver x hr) (%) 1
None DMA
2
None Craorpromazine*
1'03 x 10a 1"87 x 104
5 x 10 -3 M
98
2"51 x 105 1"44 x l0 s
2x10-3M
43
Values are means of duplicate assays. *2-Chloro-10-(3-dimethyl-aminopropyl)phenothiazine.
N,N-dimethylaniline oxidation by elasmobranch liver homogenates The ability of liver homogenates of several elasmobranchs to oxidize N,Ndimethylaniline (DMA) is shown in Table 5. DMA was readily oxidized by liver homogenates from lemon sharks and nurse sharks but to only a slight degree by liver homogenates from dogfish and skate. As shown in the table this pattern of DMA metabolism corresponds exactly to that found for the oxidation of TMA to TMAO by liver homogenates from these same species. TABLE 5--MIx~
FUNCTION (MFA) AMINE OXIDASE AND TRIMETHYLAMINE ACTIVITIES IN LIVERS OF ELASMOBRANCH FISHES
Species
Nurse shark Lemon shark Spiny dogfish Little skate
MFA oxidase
(TMA)
OXIDASE
TMA oxidase
/zmoles/g liver x hr 1-04 (2) 6"3 (2) 1.41 (4) 8.1 (4) 0"108 (2) 0"0042 (2) 0"051 (2) 0"0051 (2)
Values are averages of two to four assays (number shown in parentheses) on livers from one or two fish each.
pH optimum and substrate affinity The pH optimum and TMA Km were determined for TMA oxidase of nurse shark liver. As shown in Fig. 1, the pH optimum was approximately 9-0 in both Tris and glycine buffers. The TMA Km determined at pH 9-0 was 1.5 x 10 -4 M. DISCUSSION
Although the occurrence of trimethylamine oxide (TMAO) in vertebrate tissues has been known for many years, its biosynthesis was only fairly recently
900
LEON GOLDSTEINAND SUSAN DEWITT-HARLEY
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FIo. 1. pH activity curves of TMA oxidase of nurse shark liver. p H ' s were measured at incubation temperature--37-38°C. demonstrated (Baker & Chaykin, 1962). They showed that hog liver microsomes catalyzed the oxidation of T M A (trimethylamine) to TMAO. The enzyme was found in the livers of a number of vertebrates (Baker et al., 1963), although its occurrence in some groups, e.g. fishes, was reported to be sporadic. Thus the enzyme probably plays an important role, such as T M A detoxification, in vertebrates. The properties of the hog liver T M A oxidase reported by Baker & Chaykin are similar to those of mixed function amine oxidase (MFA), an enzyme system found in mammalian liver that catalyzes the oxidation of a number of tertiary amines to their respective N-oxides (Ziegler et al., 1969). As both enzyme systems are localized in the microsomes, require NADPH and molecular O3 for activity and are able to oxidize T M A to TMAO, it is quite possible that mammalian T M A oxidase and MFA oxidase are really the same enzyme system. Our previous studies (Goldstein & Funkhouser, 1972) on T M A oxidase in the liver of the nurse shark suggested that the enzyme in elasmobranch liver may also share common properties with mammalian MFA oxidase. If this were true then certain evolutionary implications could be derived. Therefore, we systematically determined the characteristics of T M A oxidase in nurse shark liver homogenates and compared these with the properties of mammalian MFA oxidase. As is the case with mammalian MFA oxidase, nurse shark oxidase is localized in the microsomal fraction of the liver, the enzyme system requires NADPH for activity and this requirement cannot be replaced by NADH. Neither enzyme is inhibited by inhibitors of cytochrome oxidase (azide) or P-450 (CO and SKF-525A). The pH optimum of the shark enzyme system is approximately 9.0 while that of hog liver is about 8.5. The T M A Km for the shark enzyme is approximately 0.1 raM. A T M A K m has not been reported for the hog liver MFA oxidase. However, the studies of Baker & Chaykin (1962) on T M A oxidation by hog liver microsomes indicate that the K m of the mammalian enzyme system is the same order as that
NURSE SHARK T M A
OXtD~E
901
found in the nurse shark. Thus, nurse shark T M A oxidase and hog liver M F A oxidase appear to be quite similar in all parameters measured. Further evidence for similarity of the two enzymes is the distribution of the two enzyme activities in the livers of different elasmobranchs. Livers of the two elasmobranchs that were able to oxidize T M A to T M A O were also capable of converting N,N-dimethylaniline (DMA) (a substrate of mammalian M F A oxidase) to DMAO while livers of the other two elasmobranchs examined (dogfish and skate) had only limited capability of oxidizing either substrate. The evolutionary origin and biological function of the mammalian drug metabolizing enzymes are not clear. Some of these drug metabolizing enzymes have been reported to be present in primitive classes of vertebrates such as the elasmobranchs (Adamson & Guarino, 1972), which are thought to have changed little since their first appearance approximately 400 million years ago. It is highly unlikely that the drug metabolizing enzymes evolved to detoxify the wide variety of foreign compounds that are capable of being metabolized by these enzyme systems. It is much more plausible that these enzymes evolved for different functions and coincidentally possessed the ability to detoxify foreign compounds. For example, the steroid hydroxylating enzymes which are found in both lower and higher vertebrates share a number of properties in common with the drug metabolizing enzymes--microsomal localization, NADPH requirement, CO inhibition and induction by methylcholanthrene and phenobarbital (Conney et al., 1969). Thus, it is quite likely that these enzymes evolved for the purpose of hydroxylating endogenous steroids and possessed low enough specificity to enable them to accept a number of foreign compounds as substrates. Similarly, in the present study we have shown that the enzyme system catalyzing the oxidation of T M A to T M A O in shark liver shares many properties in common with the mammalian mixed function amine oxidase. We propose that the original function of this enzyme system was the N-oxidation of T M A (and possibly other biological amines), and that the ability of the system to oxidize foreign amines is due either to a low specificity of T M A oxidase or to a secondary adaptation of the enzyme system. A more detailed comparison of the two enzymes and purification of the shark enzyme is needed before these two alternatives can be distinguished. The sporadic distribution of the T M A oxidase in elasmobranchs is puzzling. As shown in Table 6, the enzyme is found in the nurse shark, lemon shark and smooth dogfish but is absent in the spiny dogfish, little and big skates and the American stingray. There is not a clear pattern to its distribution; its occurrence does not appear to be restricted to any group of elasmobranchs or any geographical location. We had originally suggested that the enzyme might occur in elasmobranchs inhabiting tropical waters (Goldstein & Funkhouser, 1972) but its absence in the American stingray, which is found in both temperate and tropical water, weakens this theory. However, it may be noted that among the six elasmobranchs studied, the three possessing this enzyme are found in temperate and tropical waters. At any rate it is highly unusual to find such sporadic distribution of an enzyme within a single group of animals, although Adamson & Guarino (1972)
902
LEON GOLDSTEIN AND SUSAN DEWITT-HARLEY
TABLE 6--CONVERSION OF TRIMETHYLAMINE TO TRIMETHYLAMINE OXIDE IN ELASMOBRANCHS
Species Lemon shark Nurse shark Smooth dogfish American stingray Spiny dogfish Little skate
In vivo*
In vitrot
+ + + -
+ + N.T. N.T. + +
*Assayed by conversion of injected a4C-trimethylamine to a4C-trimethylamine oxide. tAssayed by conversion of 14C-trimethylamine to 14C-trimethylamine oxide in fortified liver homogenates. + , Activity present; - , activity absent; +, questionable activity; N.T., not tested.
reported similar findings for the distribution of the drug metabolizing enzyme nitroreductase in elasmobranchs. T h e enzyme was absent in the lemon shark, spiny dogfish and American stingray, but was present in the little skate. It would be interesting to determine the nature of the underlying factors controlling the distribution of T M A oxidase and other drug metabolizing enzymes in this important group of vertebrates. Acknowledgement--The authors thank Miss Anne Backus for skilful technical assistance in several phases of this study; Miss T. Devereux of the National Institute of Environmental Health Sciences for making available her procedure for assay of mixed function amine oxidase; and Dr. Ralph Miech for the sample of SKF-525A.
REFERENCES
ADAMSON R. H. & GUARINOA. M. (1972) The effect of foreign compounds on elasmobranchs and the effect of elasmobranchs on foreign compounds. Comp. Biochem. Physiol. 42A, 171-182. BAKER J. R. & CHAYKINS. (1962) The biosynthesis of trimethylamine-N-oxide. J. biol. Chem. 237, 1309-1313. BAKERJ. R., STRImMPLERA. & CHAYKINS. (1963) A comparative study of trimethylamineN-oxide biosynthesis. Biochim. biophys. Acta 71, 58-64. CONNEYA. H., LEVlN W., JACOBSONM. & KONTZMANR. (1969) Specificity in the regulation of the 6fl, 70~ and 16~-hydroxylation of testosterone by rat liver microsomes. In Symposium on Microsomes andDrug Oxidation (Edited by GILLETTEJ. R., CONNEYA. H., COSMIDES G. J., ESTABROOKR. W . , FOUTS J. R. & MANNERINGG. J.), pp. 279-295. Academic Press, New York. GOLDSTEINL. ~ FUNKHOUSERD. (1972) Biosynthesis of trimethylamine oxide in the nurse shark, Ginglymostoma cirratum. Comp. Biochem. Physiol. 42A, 51-57. GRONINGERH. S. (1959) The occurrence and significance of trimethylamine oxide in marine animals. United States Fish and Wildlife Service, Special Scientific Report. Fisheries No. 333, 1-22.
NURSE SHARK
T M A OXID~E
903
ZIEGLERD. M. & MITCHELL C. H. (1972) Microsomal oxidase--IV. Properties of a mixedfunction amine oxidase isolated from pig liver microsomes. Archs Biochem. Biophys. 150, 116-125. ZIEGLER D. M., MITCHELL C. H. & JOLLOW D. (1969) The properties of purified hepatic microsomal mixed function amine oxidase. In Symposium on Microsomes and Drug Oxidation (Edited by GmLETTE J. R., Co~q'~Y A. H., COSMIDESG. J . , ESTABROOKR. W., FOUTS J. R. & M~NERING G. J.), pp. 173-188. Academic Press, New York. ZXEG~R D. M. & PETTIT F. H. (1964) Formation of an intermediate N-oxide in the oxidative demethylation of N,N-dimethylaniline catalyzed by liver and microsomes. Biochem. biophys. Res. Commun. 15, 188-193.
Key Word Index---Trimethylamine oxidase; amine oxidase; shark liver; Ginglymostoma cirratum.