J. Biochem. 100, 77-86 (1986)

Characterization of Trimethylamine-N-Oxide (TMAO) Activity from Fish Muscle Microsomes1

Kirk L. PARKIN2 and Herbert O. HULTIN

Massachusetts Agricultural Experiment Station, Department of Food Science and Nutrition, Marine Foods Laboratory, University of Massachusetts Marine Station, Gloucester, MA 01930, U.S.A.

Received for publication, July 23, 1985

A crude microsomal fraction isolated from red hake (Urophycis chuss) muscle de- methylated trimethylamine-N-oxide (TMAO). Two systems were capable of stimulating activity; the system of NADH and FMN required anaerobic condi tions while the other system, composed of iron and cysteine and/or ascorbate func tioned in the presence or absence of oxygen. The components of each cofactor system functioned synergistically and kinetic parameters were established for each. Of several amine compounds common to fish muscle, TMAO was the only substrate demethylated by the microsomes. Activity was inhibited by iodoacetamide, potas sium cyanide, and sodium azide under certain conditions, but not by carbon mon- oxide. An enzymic nature of the reaction was demonstrated by the properties of heat lability, sensitivity to protease treatment, the requirement of microsomes for TMAO demethylation and by the exhibition of typical hyperbolic kinetics with respect to substrate (TMAO). Moreover, TMAO demethylation by the microsomes was 3 to 4 orders of magnitude faster than the non-enzymic reaction and the reac tion was specific for dimethylamine (DMA) as product. It appears the two cofactor systems may share a common catalytic unit in the process of TMAO demethylation.

The ability of subcellular fractions of hepatic liver tissue (2-4). Three of hepatic mi tissue to catalyze the N-dealkylation of tertiary crosomes that produce formaldehyde (HCHO) are amines was noted several decades ago (1). Many amine oxidase, catalase, and cytochrome P-450 dealkylating systems lead to the liberation of form- (5) and these accept a broad range of substrates (demethylation) and some of these (6, 7). Horseradish peroxidase is also capable of reactions are recognized as mediating the biotrans catalyzing N-demethylation reactions (8) and in formation of xenobiotic compounds, especially in mitochondria, HCHO is generated from betaine

1 This work was supported in part by the Massachusetts Agricultural Experiment Station , by the Graduate School of the University of Massachusetts at Amherst, and by NOAA and the New England Fisheries Development Foundation. K.L.P. was the recipient of a Ralston-Purina Graduate Fellowship. A portion of this work was presented at the meeting of the American Chemical Society, Washington, D.C., August, 1983. 2 Present address: Department of Food Science , University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

Vol. 100, No. 1, 1986 77 78 K.L. PARKIN and H.O. HULTIN

and sarcosine by an electron transfer reaction sys- tained from Eastman. Sarcosine, phenylmethyl tem (9). sulfonyl fluoride (PMSF), flavin mononucleotide

Hepatic microsomal demethylating systems (FMN), bovine serum albumin (BSA) fraction V, have been purified and characterized. Microsomal type ‡\ glucose oxidase (GOx), and grade III mixed function amine oxidase catalyzes the oxida flavin adenine dinucleotide (FAD) were purchased tion (10) and the dealkylation (11, 12) of several from Sigma. Grade I reduced nicotinamide ade tertiary amines and amine oxides. These two nine dinucleotide (NADH) and reduced nicotin discreet activities have been characterized by sub amide adenine dinucleotide phosphate (NADPH) strate specificity, cofactor requirements, and in were products of Boehringer-Mannheim. Benzene hibitor sensitivities (10, 13). Study of the reac was spectrophotometric grade. All other chem tions catalyzed by cytochrome P-450 have often icals were reagent grade or the best grade avail- been conducted with microsomal particles since able commercially.

many of the P-450 reactions are interrrelated with Preparation of a Crude Microsomal Fraction an intact and complex membranous electron trans -Red hake muscle microsomes were prepared as

fer system (6). previously described (23) except that the blending Certain species of bacteria have the capability step was replaced by mincing the muscle with an

of demethylating methylamine carbon sources (14). electric meat grinder. Minced muscle (held in

Assimilation of C, fragments by these methyl ice) was homogenized in chilled 0.12 M KCl, 5 mm otropic organisms involves a variety of inducible histidine, pH 7.3 with a Brinkmann Polytron ho enzymes (15), one of which is a trimethylamine-N- mogenizer at a rheostat setting of 60. Homogeni oxide (TMAO) demethylase (16-18). This zation was accomplished by two 45 s or 1.5 min

has commonly been studied as a crude, cell-free bursts for 100 or 200 g of minced muscle, respec sonic extract (16, 18, 19), although purification to tively, that had been mixed with 3 to 4 times its homogeneity has also been accomplished (17). The weight in volume of buffer. The homogenate was enzyme from various bacterial sources appears to centrifuged at 21,460 x gmax for 30 min. The re

have different characteristics and it has not been sulting supernate was filtered through a double reported to be associated with any subcellular layer of cheesecloth and centrifuged at 104,400 fraction. x gmax for 60 min. The resulting pellet was We have previously reported the ability of a washed by suspending in 0.6 M KCl, 5 mm histi

fish muscle microsomal fraction to catalyze the dine, pH 7.3, and homogenizing by 5 strokes with demethylation of TMAO (20). There have been a Potter-Elvehjem tissue homogenizer. After sit- few reports characterizing this enzyme from fish, ting on ice for 60 min, the suspension was recen

and these have been confined to preparations from trifuged at 104,000 x gmax for 60 min. The re liver (21) and kidney (22) tissue. A detailed char sulting pellet was resuspended in 0.24 M KCl, 10

acterization of TMAO demethylase activity in fish mM histidine, pH 7.3 by 10 strokes with a Potter- muscle may provide insight as to its physiological Elvehjem tissue homogenizer, and this constituted function. The purpose of this work was to estab the crude microsomal preparation. This prepara lish some of the kinetic properties of TMAO de tion for microsomal membranes will be referred to methylase associated with muscle microsomes. as MI. When it was found that TMAO demethyl

ase activity was most enriched in a light microsomal

MATERIALS AND METHODS fraction sedimenting between 104,400 and 142,100 X gmax, subsequent preparations were obtained by

Fish-Fresh red hake were procured from day replacing the 104,400 x gmax centrifugation steps boats in Gloucester, MA and transported to the with ones at 142,100 x gmax. This preparation will

laboratory on ice. The fish were filleted and held be referred to as M‡U. It was found that EDTA,

at -80°C until use whereupon they were thawed mercaptoethanol and PMSF enhanced yield and

in water at 10-15°C. stability of microsomal TMAO demethylase activ

Reagents-Trimethylamine-N-oxide (TMAO), ity when included in the preparative buffers. When

betaine, choline, dimethylamine (DMA), trimethyl added to the preparative buffers, their final con amine (TMA), and iodoacetamide (IAA) were ob centrations were 0.2 mm PMSF (from 0.2 M stock

J. Biochem. FISH MUSCLE MICROSOMAL TMAO DEMETHYLATION 79

in isopropanol; final isopropanol concentration of DMA analysis by the copper dithiocarbamate 0.1 % in preparative buffer); mercaptoethanol, 2 procedure (24) with shaking accomplished by a mm; and EDTA, 2 mm in the 0.12 M KCl , 5 mm Cole-Palmer roto-torque (Model 7637) at maximal histidine buffer and 0.05 mm in the 0.6 M KCl, 5 speed. mm histidine buffer. Microsomal preparations Activity as HCHO Formation-The activity isolated as Mr and M‡U in the presence of these of incubating mixtures was stopped by the addi stabilizing agents will be referred to as Mt* and tion of 1 % HCl in absolute ethanol at a ratio of M‡T*, respectively. 2 ml acid-alcohol per ml of incubation mixture. Determination of TMAO Demethylase Activity After clarification of the acid-alcohol extract by -Activity of the crude microsomal and other sub- centrifugation for 10 min at 2,000 x gm,, an ali cellular fractions was determined in chromic acid- quot of the extract was assayed for HCHO by the washed flasks. All reaction components were tryptophan-FeCl3-sulfuric acid method (25). Zero- added to the flasks and the enzymic reaction ini time (blank) samples were prepared for each assay tiated by the simultaneous addition of TMAO and condition, as were blanks for samples without membranes. Non-enzymic activity of mixtures of microsomes and without TMAO. components was initiated by the addition of TMAO Protein Determination-Protein was measured alone. Anaerobic conditions were obtained by by a modified Lowry method (26) with BSA used GOx and glucose at 9 U/ml and 13 mm, respec as the standard. tively, in the reaction medium (verified by oxygen polarographic measurement; none of the compo RESULTS nents included in the reaction mixtures significantly

affected GOx activity). Incubations were con Subcellular Fractionation of Muscle Tissue and

ducted at 20°C in a shaking water bath for 30 min. Location of TMAO Demethylase Activity-TMAO

A linear rate of DMA formation for at least 30 demethylase activity was observed in all crude sub-

min was confirmed under all conditions employed. cellular fractions of red hake muscle (data not

The reaction was terminated by the addition of shown). Activity in the presence of the two dis

acid or acid-alcohol mixtures, depending on the creet cofactor systems became most enriched in

assay method used. Under all assay conditions the crude fractions sedimented between 30K-

examined, DMA or HCHO formation in the 142K x gmax for 60 min and in each case con

absence of membranes was < 1 % of that produced stituted 14% of the original activity of the initial

in the presence of membranes (and in most cases muscle homogenate. The specific activity of this

not detected) under identical conditions. All as- crude microsomal fraction was 2.12 t

says were performed in duplicate and activities are mg protein • h for activity catalyzed by iron, cys

reported as the means ±S.D. There was sometimes tein and ascorbate and 6.95 ƒÊmol DMA/mg pro

considerable variation in the specific activity of tein • h for activity catalyzed anaerobically by FMN

different microsomal preparations. For this rea and NADH, representing a fold purification of son, the data is presented following normalization 28.3 and 29.1, respectively compared to the cor-

to 100% for the conditions yielding the greatest responding specific activities of the homogenate. specific activity for that series of experiments. In Whether the presence of activity in all fractions

addition, the range of specific activities found for was due to contamination by the membrane sys-

the various preparations used for the experiments tem specific for activity was not investigated.

is provided in the table and figure legends. Fifteen percent of the activity recovered from Activity as DMA Formation-DMA and homogenized muscle remained `soluble' (not sedi

HCHO are produced in equimolar amounts from mented at 142,100 x gm,, in 60 min). the enzymic demethylation of TMAO (17, 21). Characterization of Crude Microsomal TMAO Incubations were stopped with 25 % trichloroacetic Demethylase Activity-Crude microsomes demeth- acid (TCA) at a ratio of I ml TCA to 4 ml reaction ylated TMAO in the presence of either of two mixture. The acidified mixture was then vortexed cofactor systems (Table I). Under the conditions for 5 s and centrifuged at 2,000 x gmax for 10 min. investigated, stimulation of activity by FMN and An aliquot of the cleared extract was subjected to NAD(P)H was about 5-fold greater than that by

Vol. 100, No. 1, 1986 80 K.L. PARKIN and H.O. HULTIN

TABLE I. Production of DMA by crude muscle micro Activity was linear with respect to microsomal somes. Concentration of the components were 20 mm protein concentration up to at least 10 mg protein TMAO, 2 mm ascorbate, 2 mm cysteine, 0.2 mm FeCl2, per ml reaction mixture (data not shown). A 30 0.1 mm FMN, 0.4 mm NADH and assay conditions were min pre-treatment of crude microsomes with a 0.12 M NaCl, 25 mm histidine, pH 7.0 at 20°C. All non-specific bacterial protease (33 jig Sigma Pro samples contained microsomes (prepared as MI or M‡U*) nase E per mg protein) at 0°C resulted in solu at 0.70 to 2.0 mg protein/ml and 100% activity ranged from 3.67 to 4.74 ƒÊmol DMA/mg.h for 3 experiments. bilization of 56% of the membrane protein and a 48 % loss of demethylase activity. The microsomes were incapable of demethylat ing sarcosine, betaine, choline, TMA, DMA (all at 20 mm), and dimethylaniline at saturation when assayed for HCHO with either cofactor system. However, an equimolar production of DMA and HCHO was observed for activity on TMAO as 0.95 •} 0.04 and 0.88 •} 0.11 ƒÊmol of HCHO and DMA, respectively, were formed per mg protein.h for activity stimulated by iron, cysteine, and ascor bate, and 7.50•}0.37 and 8.16 •} 1.40 ƒÊmol of HCHO and DMA, respectively, were formed per mg protein.h for activity caused anaerobically by FMN and NADH.

Microsomal demethylase activity as a func

tion of the concentration of the individual compo

nents of each cofactor system was investigated.

It was previously noted that for the ascorbate,

cysteine, and iron system, TMAO demethylase ac

tivity in the microsomes was linear and non-

ascorbate, iron and cysteine. Either NADHH or preferential with respect to FeCl2 or FeCl3 (20). NADPH could be used with similar efficiency, In this study, no activity was observed when iron

whereas FMN was preferred over FAD as the was replaced by the chloride salts of Ca 2+' Mn2+,

flavin moiety. Activity was observed in the pres Mg2+, Nit+, Zn2+, or Co2+ under identical condi- ence of FMN or NADH alone, and stimulation by tions for the ascorbate, cysteine, and iron cofactor

these components was synergistic when both were system. Microsomal activity as influenced by present. Activity generated by fiavins and pyri ascorbate and cysteine concentration was reported dine nucleotides required anaerobic conditions. earlier (20). Microsomal activity as a function A synergistic relationship was also observed for of NADH concentration in the presence of FMN microsomal activity stimulated by ascorbate and and the absence of oxygen is presented in Fig. 1. cysteine in the presence of iron; maximal activity TMAO demethylation appears to be slightly sig was generated when both of these components moidal with respect to NADH concentration in were included with iron in the assay mixture. the presence of FMN. Similarly, activity as a Activity with these cofactors was independent of function of FMN in the presence of NADH and the presence or absence of oxygen. An important absence of oxygen displayed sigmoidal kinetics

control condition showed that microsomes and (Fig. 2). The lowest concentration at which ac FeCl2 alone under anaerobic conditions (generated tivity was initially stimulated by either NADH or by GOx/glucose) did not demethylate TMAO. The FMN varied from 10 to 50 ƒÊM among the various microsomal fraction lost all activity catalyzed by microsomal preparations. both cofactor systems upon heating at 100°C for Microsomal activity as a function of TMAO 5 min and when either set of cofactors and TMAO concentration with each of the cofactor systems is were incubated in the absence of microsomes, no presented in Fig. 3. Both systems display satura DMA or HCHO was formed (data not shown). tion kinetics with respect to TMAO. Eadie-

J. Biochem. FISH MUSCLE MICROSOMAL TMAO DEMETHYLATION 81

Fig. 3. Microsomal activity as a function of TMAO

Fig. 1. Microsomal activity as a function of NADH concentration. Assay conditions were 0.12 M KCl, 50 concentration. Assay conditions were anaerobic with mm histidine, pH 7.0 at 20°C and I to 2 mg protein

0.1 mm FMN, 50 mm TMAO, 0.12 M NaCl, 25 mm (Mr)/ml. The closed circles represent activity catalyzed histidine, pH 7.0 at 20°C and 0.6 to 0.8 mg protein aerobically by 2 mm ascorbate, 2 mm cysteine and 0.2

(M‡U*)/ml. Values are expressed as the mean values mm FeCl2 where 100% activity ranged from 0.064 to from 4 experiments where 100% activity ranged from 0.28 ƒÊmol DMA/mg•Eh for 3 experiments. The open 0.54 to 6.40 ƒÊmol DMA/mg-h. Inset has the same circles represent activity catalyzed anaerobically by 0.1 axes and highlights the effect of low NADH concen mm FMN and 0.4 mm NADH where 100%, o activity trations. ranged from 1.62 to 2.62 ƒÊmol DMA/mg-h. Values

are expressed as the means±S.D. Inset has the same axes and highlights the effect of low TMAO concen trations.

Fig. 2. Microsomal activity as a function of FMN concentration. Assay conditions were the same as Fig. 4. Thermal deactivation of microsomal activity. reported in Fig. I except that FMN concentration was The microsomal suspension was heated at 40°C in varied and 0.5 mm NADH was included as a constituent 0.12 M KCl, 25 mM histidine, pH 7.0. Assay conditions

of the reaction medium. Values are expressed as the were 2 mm ascorbate, 0.4 mat FeCl2, 50 mm TMAO, mean values from 4 experiments where 100% activity 0.12 M KCl, 25 mm histidine, pH 7.0 at 20"C and 1.5 mg ranged from 0.76 to 7.16 ƒÊmol DMA/mg.h. Inset protein (Mr)/ml where 100° activity was 0.084 /ƒÊmol has the same axes and highlights the effect of low FMN DMA/mg.h for a single experiment. concentrations. (2 mm) and anaerobieally by FMN (0.1 mm) and Scatchard plots (not shown) and analysis by linear NADH (0.4 mm), respectively. regression yielded Km values for TMAO of 2.7 Deactivation of microsomal demethylase ac mm (range of 2.4 to 3.0 mm) and 21 mm (range of tivity occurred during incubation at 40`C (Fig. 4). 14 to 32 mm) for the reaction catalyzed aerobically The kinetics of deactivation were complex in that by iron (0.2 mm), ascorbate (2 mm), and cysteine simple first-order rate laws did not apply.

Vol. 100, No. 1, 1986 82 K.L. PARKIN and H.O. HULTIN

Inhibitors of Microsoinal TMAO Denzethylase anaerobic conditions, whereas activity was un- Activity-The susceptibility of demethylase activ affected by azide in the presence of oxygen (data ity to inhibitors is presented in Table II. Micro not shown). somal activity stimulated by either cofactor system Components of one cofactor system were was inhibited equally (about 75%) by KCN and found to inhibit activity catalyzed by the other in virtually unaffected by CO. In addition, pre-incu some cases (Table III). In the presence of 0.2 bation of the microsomes with iodoacetamide re mm FeCl2, activity stimulated by FMN and NADH sulted in a loss of 25 % of the activity generated was inhibited by 95 %. The activity observed under by both cofactor systems. A major difference was these conditions was similar to that generated when observed between cofactor systems in response to FeCl2 and ascorbate alone were added to the mi azide. Activity catalyzed anaerobically by FMN crosomes. Ascorbate inhibited 40% of the activ- and NADH was essentially completely eliminated in the presence of azide whereas activity generated TABLE III. Effect of components of one cofactor by ascorbate, cysteine, and iron was inhibited by system on activity stimulated by the other. Assay condi less than 50% anaerobically and not at all in the tions and concentrations of components were the same presence of oxygen. A similar trend was observed as reported in Table I. One hundred % activity ranged for activity stimulated by cysteine and iron or from 0.61 to 1.24 ƒÊmol DMA/mg. h for Control A and ascorbate and iron ; approximately 50 % inhibition 1.40 to 5.75 ƒÊmol DMA/mg.h for Control B for 3 ex was observed in the presence of azide only under periments.

TABLE II. Inhibitors of microsomal activity stimu lated by FMN and NADH or by ascorbate, cysteine, and iron. Assay conditions and concentrations of the components were the same as reported in Table I; KCN and azide were 1 mm. One hundred activity ranged from 0.14 to 0.93 i mol DMA/mg.h for Control A and

1.40 to 5.75 ƒÊmol DMA/mg.h for Control B for 2 ex periments.

a CO was slowly bubbled into a microsomal suspension at 0°C for 10 min prior to assay. b Pre-treatment of a microsomal suspension with 5 inst IAA was for 30 min at 0°C followed by recentrifugation and resuspension in assay buffer prior to assay.

J. Biochem. FISH MUSCLE MICROSOMAL TMAO DEMETHYLATION 83

ity stimulated by FMN and NADH; inhibition 17 mm, respectively); under these conditions the in the presence of both ascorbate and FeCl2 was concomitant formation of 2 mm TMA takes place intermediate between the effects of the individual (27). Calculating data generated by others, non- components. The inhibition by FeCl2 could not enzymic demethylation of TMAO (27 mm) in the

be relieved by the presence of EDTA. presence of aqueous Fe2+ (1 mm) yields about 0.01 Under some conditions FMN and NADH ex mm DMA over a 24 h period, or 0.42 ƒÊM/h (28). erted an inhibitory effect on microsomal activity A typical rate of the microsomal-catalyzed reaction

stimulated by ascorbate, cysteine, and iron (Table observed in this study in the presence of 0.2 mm III). For activity caused by FeCl2 and cysteine, Fe_+ and 20 mm TMAO was about 0.35 mm DMA/ the presence of FMN was inhibitory while NADH h. (For this latter calculation, the value of 4.0 had no significant effect. The presence of NADH ƒÊ mol DMA/mg-h was taken as 100% activity and and/or FMN inhibited activity brought about by the rate of ascorbate-FeCl2-catalyzed reaction was ascorbate and FeCl2. In the presence of FeCl2, used assuming a protein concentration of 1 mg/ ascorbate, and cysteine, FMN, but not NADH, ml; this yields a rate of 0.35 ƒÊmol DMA/mg-h or inhibited TMAO demethylation. 0.35 mm DMA/h-refer to Table I.) Thus, the

rate of the microsomal-catalyzed DMA production

DISCUSSION is roughly 3 orders of magnitude greater than that calculated for the non-enzymic reaction with a

Although it has been shown that TMAO demeth 5-fold higher [Fe -+] in the latter case. Making a

ylation can take place in the presence of iron and similar calculation for the reaction stimulated

cysteine via non-enzymic means (27-29), our crude anaerobically by NADH and FMN yields a rate microsomal TMAO demethylating system appears of DMA formation of 4 mm DMA/h, some 4

to be enzymic. TMAO demethylation took place orders of magnitude greater than that for the

only in the presence of the membranes and upon non-enzymic reaction. These enhanced reaction

heating at 40 C, activity generated by both co- rates are indicative of the enzymic nature of the factor systems was lost. Physical changes brought microsomal demethylating system. Moreover, we

about in the membrane during heating may, how- observed negligible amounts of TMA formed

ever, account for some of the loss in activity. The (restricted to at most, 1.7% of the DMA produced) proteinaceous nature of the demethylase system is by reactive mixtures of microsomes and cofactors. supported by its sensitivity to pre-incubation with This highlights the specificity of product formation a non-specific protease and the observation that by the microsomal reaction; TMAO decomposi membrane isolation in the presence of PMSF re tion to DMA by non-enzymic means is accom

sulted in enhanced yield and stability of activity. panied by concomitant formation of TMA (27, 29), The utilization of substrate by the muscle micro with the latter being the principal product of the somes displayed typical Michaelis-Menten enzyme reaction at ambient temperatures (27). It is un- kinetics when either cofactor system was employed. likely that microsomal TMAO demethylation

In a related study (30), we have found that a proceeds via reduction to TMA, followed by lysis partially purified enzyme prepared by detergent to DMA and HCHO; TMA is not found in sig treatment displayed substrate inhibition, a feature nificant amounts in reacting microsomal suspen not commonly associated with non-enzymic reac sions, it does not serve as a substrate for demethyl tions. ation, and it inhibits the rate of TMAO demethyl The non-enzymic reaction of TMAO with ation. Over the range of 0 to 20•Ž, the E,, for iron and reductant yields TMA and DMA at a the microsomal demethylase reaction was deter- molar ratio of 20 : 1 at pH 7.0 and 20•Ž (27). mined to be 15.7 and 5.6 kcal/mol as stimulated

At 200`C, non-enzymic decomposition of TMAO by NADH/FMN and ascorbate/cysteine/FeCl2, in squid tissue yields roughly equirnolar amounts respectively. These values are compatible with of TMA and DMA (29). Significant non-enzymic those normally observed for enzymic reactions; it DMA formation (calculated as ca. 0.1 mM/h was not possible to calculate an En, for the studies from 8.3 mm TMAO) is only observed when high on the non-enzymic demethylation reaction for levels of FeCl2 and cysteine are used (0.85 mm and comparison.

Vol. 100, No. 1, 1986 84 K.L. PARKIN and H.O. HULTIN

Although we used the GOx/glucose system to what was observed in this study for TMAO for generating the anaerobic conditions required demethylation by red hake muscle microsomes (2.7 for activity by FMN and NADH, activity under mm) under the conditions employed. Enzymic anaerobic conditions was not mediated by H2O2 TMAO demethylation by a crude fish liver (32) production by GOx. The preparation of this en preparation is also stimulated by iron and ascor zyme was enriched 4-fold with catalase which was bate (in the presence of methylene blue). capable of reducing the levels H.,Oz in the incubat Red hake muscle microsomal TMAO demeth ing mixtures. Microsomes incubated in the pres ylation was also stimulated anaerobically by flavins ence of TMAO under anaerobic conditions gen and pyridine dinucleotides. A similar requirement erated by GOx/glucose were not reactive without for activity has been observed by a crude soluble NADH and FMN. Activity with the iron-reduc enzyme preparation from muscle tissue of this fish tant system also does not appear to be mediated species (31). Hepatic microsomal NADPH-cyto by a metal-catalyzed active oxygen mechanism. chrome P-450 reductase is a flavoprotein composed These cofactors in the absence of microsomes did of FAD and FMN that is responsible for reducing not demethylate TMAO under the conditions em P-450 (33), and NAD(P)H is also required for ployed and the addition of catalase and superoxide demethylation reactions catalyzed by P-450 (3, 4) dismutase had no effect on the demethylase activity and mixed function amine oxidase (10). NADPH of incubating mixtures (20). Most importantly, is preferred over NADH for these latter two en when microsomes were incubated with TMAO and zyme systems. However, no preference was ob- FeCl2 under anaerobic conditions (GOx/glucose), served for either of these pyridine dinuleotides for no DMA formation was observed. Thus, the pos demethylase activity by fish muscle microsomes. sibility that a H2O2-FeCl2-mediated active oxygen Another difference between the hepatic and fish mechanism for TMAO demethylation can be dis muscle microsomal demethylation reactions is that counted. In addition, microsomes incubated with the former requires oxygen for activity (3, 4) iron and TMAO without reductant failed to pro whereas the latter was inhibited by oxygen when duce DMA, in spite of extensive lipid oxidation the reaction was catalyzed by FMN and NADH. taking place (high TBA number) with the likely The bacterial enzyme can demethylate other involvement of activated oxygen species. Others amine oxides at rates no greater than 15 % of the have concluded that TMAO in the presence of activity observed with TMAO (17). Hepatic mi autoxidizing lipids, does not decompose non- crosomes can demethylate a variety of amines, enzymically to form DMA (28). Also, TMAO amine oxides and other xenobiotic compounds (2, demethylation by an cytosolic enzyme from hake 6, 7). These demethylating systems may be ex muscle was observed with these same cofactor pected to act on a variety of substrates due to systems; whether anaerobiosis is generated by their respective physiological functions of carbon evacuation in Thunberg tubes or by the GOx/ assimilation and detoxification. We did not fully glucose mixture had no effect on activity (31). survey substrate specificity, but found the fish Two discreet cofactor systems were capable of muscle microsomes to demethylate only TMAO stimulating TMAO demethylation by a crude mi when this and some amines common to fish muscle crosomal fraction prepared from red hake muscle. were tested as substrates. Other studies demon Activity stimulated by iron required either ascor strated that a crude enzyme preparation from fish bate or cysteine and could function in the presence pyloric caeca could demethylate dimethylaniline- or absence of oxygen. The requirements for mi N-oxide at a rate 6 % of that observed for TMAO, crosomal activity by this cofactor system are simi- while TMA, betaine, choline, and DMA could not lar to those for TMAO demethylation by a sonic serve as substrates (32, 34). An earlier observation extract of Bacillus PM6 (16, 17). For the bacterial that TMA and choline inhibited TMAO demeth preparation, TMAO demethylation requires iron, ylation (20) may imply the importance of the tri ascorbate and a thiol compound in the presence methylated amine structure in membrane/enzyme- or absence of oxygen, although generally, the reac substrate recognition. The significance of the tion rate is faster under anaerobic conditions. The TMAO demethylase system in fish muscle is not Kr„ for the bacterial enzyme (2.85 mm) is similar known although there may be a need for marine

J. Biochem. FISH MUSCLE MICROSOMAL TMAO DEMETHYLATION 85

organisms to regulate tissue concentrations of amide inhibited activity by both cofactor systems TMAO (35). by about 25 %. An earlier study found that Cue An unusual pattern was previously noted for was also an effective inhibitor of activity (20) and ascorbate stimulation of TMAO demethylation by the inclusion of mercaptoethanol in the preparative the muscle microsomes (20). This biphasic pat- buffers enhanced recovery and stability of micro tern could be explained by the existence of two somal activity. These observations may indicate mechanisms for activity, one operating at low the importance of sulfhydryl groups for demethyl ascorbate concentrations and the other at high. ase functioning. Alternatively, ascorbate stimulation may be medi ated by two different protein components in the REFERENCES membrane. This latter situation may be indicated 1. Fish, M.S., Johnson, N.M., Lawrence, E.P., & by the observation that heat deactivation at 40°C Horning, E.C. (1955) Biochem. Biophys. Acta 18, of microsomal activity did not fit a first-order plot. 564-565 The rate of TMAO demethylation by the mi 2. Mason, H.S., North, J.C., & Vanneste, M. (1965) crosomes is linear with respect to iron concentra Fed. Proc. 24, 1172-1180 tion and shows no preference for either ionic (2+ 3. Estabrook, R.W. & Cohen, B. (1969) in Microsomes or 3+) state (20). Stimulation by iron was cata and Drug Oxidations (Gillette, J.R., Conney, A.H., lytic as there was often more reaction product Cosmides, G.J., Estabrook, R.W., Fouts, J.R., & observed than the amount of iron present in the Mannering, G.J., eds.) pp. 95-105, Academic Press, assay medium. It was concluded that iron func Inc., New York 4. Chambers, J.E. & Yarbrough, J.D. (1976) Comp. tions through a cycling mechanism between the Biochem. Physiol. 55C, 77-84 ferrous and ferric states. The requirement of 5. Werringloer, J. (1978) in Methods in Enzymology ascorbate or cysteine for activity is most likely as (Fleischer, S. & Packer, L., eds.) Vol. 52, pp. 297- an external ferric iron reductant. The bacterial 302, Academic Press, Inc., New York demethylase also utilizes iron in a catalytic, redox 6. Estabrook, R.W. (1978) in Methods in Enzymology cycling mechanism (17). Similarly, P-450-catalyzed (Fleischer, S. & Packer, L., eds.) Vol. 52, pp. 43- hydroxylations involve a catalytic ferrous-ferric 47, Academic Press, Inc., New York transition (6). However, in this latter case, the 7. Wislocki, P.G., Miwa, G.T., & Lu, A.Y.H. (1980) iron constitutes a portion of the prosthetic group, in Enzymatic Basis of Detoxification (Jakoby, W.B., whereas in the former examples iron is exogenous ed.) Vol. 1, pp. 135-182, Academic Press, Inc., to the enzyme. New York It appears that a common catalytic unit may 8. Renneberg, R., Damerau, W., Jung, C., Ebert, B., & Scheller, F. (1983) Biochem. Biophys. Res. Conmmun. be shared by both cofactor systems catalyzing the 113,332-339 demethylation of TMAO. In the course of sub- 9. Frisell, W.R., Cronin, J.R., & MacKenzie, C.G. cellular fractionation, activity stimulated by these (1966) in Flavins and Flavoproteins (Slater, E.C., ed.) cofactor systems segregated to similar extents in pp. 367-387, Elsevier, New York each of the subfractions. Also, there was an 10. Ziegler, D.M. & Pettit, F.H. (1966) Biochemistry antagonistic relationship between the two cofactor 5,2932-2938 systems, implying competition for a single site. 11. Pettit, F.H. & Ziegler, D.M. (1963) Biochem. Bio Furthermore, inhibitor sensitivities for activity phys. Res. Common. 13, 193-197 stimulated by both cofactor systems are similar 12. Terayama, H. (1963) Gann 54, 195-204 with CO having no effect and KCN and azide 13. Machinist, J.M., Orme-Johnson, W.H., & Ziegler, D.M. (1966) Biochemistry 5, 2939-2943 (anaerobically) both inhibitory. This implies the 14. Large, P.J. (1971) Xenobiotica 1, 457-467 participation of a heme moiety in the reaction; in 15. Colby, J. & Zatman, L.J. (1973) Biochem. J. 132, subsequent studies we have demonstrated that both 101-112 of these cofactor systems are capable of reducing 16. Myers, P.A. & Zatman, L.J. (1971) Biochem. J. a muscle microsomal hemoprotein that can be 121,10 reoxidized by TMAO under certain conditions 17. Myers, P.A. (1971) Ph.D. Dissertation, University (Parkin, K.L. & Hultin, H.O., unpublished results). of Reading Pre-incubation of the membranes with iodoacet- 18. Large, P.J. (1971) FEBS Lett. 18, 297-300

Vol. 100, No. 1, 1986 86 K.L. PARKIN and H.O. HULTIN

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