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Reduction of p-nitrobenzoic acid by fishes
ARCHIVES
OF
BIOCHEMISTRY
AND
Reduction
BIOPHYSICS
103,
and Wildlife, Received
(1968)
of p-Nitrobenzoic
DONALD R. BUHLER2 Bureau of Sport Fisheries
.%?-,%j
May
AND
Fish-Pesticide
Acid
by Fishes’
MARY E. RASMUSSONa Research Laboratory,
25, 1967; accepted
September
Columbia,
Missouri
66.%‘01
7, 1967
A system which catalyzes the reduction of p-nitrobenzoic acid to p-aminobenzoic acid has been found in a number of freshwater fishes. The nitro reductase system occurred primarily in the liver, where it was associated with the soluble fraction of the cell. Fish liver nitro reductase required NADPH and was inhibited by oxygen. Preincubation of the system in air caused a loss of nitro reductase activity, suggesting that some sensitive component of the system had undergone oxidation. Some evidence was advanced relating this inactivation with the formation of lipid peroxides. Various sulfhydryl compounds and chelating agents could reverse or prevent the oxygen-derived inactivation. Additional evidence indicated that the fish liver nitro reductase system contained essential sulfhydryl groups and a carbon monoxide-sensitive component.
Aromatic nitro compounds are reduced under anerobic conditions to the corresponding amines by mammalian tissue homogenates (l-3). The enzyme responsible for these reductions occurs primarily in the liver in both the soluble fraction of the cells and in microsomes (2). Both NADPH and NADH can serve as electron donors for the mammalian nitro reductase system; however, NADPH is considerably more effective. The mammalian system is almost inactive in the presence of oxygen. Mammalian nitro reductase includes a flavoprotein component and is greatly stimulated by addition of excess flavin. A possible relationship between nitro reductase and the flavoprotein enzyme NADPH-cytochrome c reductase has been proposed by Kamm and Gillette (4), and a more recent study has suggested that 1 These investigations were carried out at the Western Fish Nutrition Laboratory, Cook, Washington. A portion of this work was presented at the annual meeting of the Federation of American Societies for Experimental Biology in Atlantic City, New Jersey, April, 1966 [Federation Proc. 26, 343 (1966)]. 2 Present address: Department of Agricultural Chemisty, Oregon State University, Corvallis, Oregon 97331.
cytochrome P-450 is a component of the mammalian nitro reductase system (5). Recently, Adamson and co-workers (6) detected azo and nitro reductase activity in the liver of two species of teleost fish. Hitchcock and Murphy (7), in addition, have recorded the reduction of nitro groups in several organic phosphorus insecticides by fish tissues. The present studies demonstrate the occurrence of an NADPH-dependent nitro reductase system in a number of fish species. The enzyme system is found mainly in liver tissue, where it is associated with the soluble fraction of the cell. The enzyme from fish liver has been characterized and found to be similar in many respects to mammalian nitro reductase, but it differs in its absence from the microsomal fraction. MATERIALS
AND
METHODS
Animals. Bluegill (Lepomis macrochirus), white crappie (Porno&s annularis), and yellow perch (Perca javescens) were obtained from a nearby lake on a hook and line. Carp (Cyrpinus carpio), Pacific lamprey (Lampetra tridentala), sockeye salmon (Oncorhynchus nerka), American shad (Alosa sapidissima), white sturgeon (Acipenser transmontanus), largescale sucker (Calostomus 582
REDUCTION
OF p-NITROBENZOIC
macrocheilus), and steelhead trout (Salmo gairdneri gairdneri) were trapped from the Columbia River at the Fish Passage Laboratory,8 U.S. Bureau of Commercial Fisheries, North Bonneville, Washington. Smallmouth bass (Micropterus dolomieui) , channel catfish (Zctalurus punctatus), flathead catfish (Pylodictis olivaris), and northern pike (Esoz Z&us) were obtained from the Miles City National Fish Hatchery, Miles City, Montana. Fingerling coho salmon (0. kisutch) and adult rainbow trout (S. gairdneri) were stock fish of the Western Fish Nutrition Laboratory, Cook, Washington. Fish were transported to the laboratory alive and maintained in large tanks until used experimentally. Freshly killed adult chinook salmon (0. tshawytscha) and coho salmon were collected immediately after spawning at the Spring Creek and Little White Salmon National Fish Hatcheries,’ Cook, Washington, and appropriate tissue samples were quickly transferred to the laboratory in cold isotonic KCl. Enzyme preparation. Fishes were exsanguinated, and livers and other tissue were removed immediately and homogenized in 2 volumes of cold 0.154 M KC16 with a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 10,OOOg for 20minutes, and the supernatant fractions, contain ing microsomes plus soluble fraction, were used for the majority of thestudies reported. Washedmicrosomes were prepared by centrifugation of the 10,OOOgsupernatantfractionat 105,OOOg for 60 minutes. After removal of the supernatant soluble fraction, the sedimented microsomes were suspended in fresh KCl, recentrifuged, and then resuspended in the original volume of 0.154 M KCl. Enzyme assays. Various fish enzyme preparations were incubated in duplicate with p-nitrobenzoic acid in a Dubnoff metabolic shaking incubator, usually for 2 hours at 25”. In most experiments the incubation mixture contained 1 ml of a tissue fraction derived from 0.34 gm fresh tissue, 0.5 Mmole NADP,” 1.25 rmoles MgCh, 10 rmoles glucose 6-phosphate, 1 Kornberg unit of glucose g-phosphate dehydrogenase (or 10 rmoles sodium isocitrate and 2 mg of isocitric dehydrogenase), 3 3 The cooperation of the late Mr. Joseph Cauley and his staff is gratefully acknowledged. 4 Arranged through the cooperation of Mr. Harlan Johnson. 5 Homogenization in potassium phosphate buffer, sucrose, or Tris-sucrose-KC1 gave preparations with somewhat lower nitro reductase activity. 6 Cofactors, enzymes, and additives to the incubation mixtures were products of the Sigma Chemical Co., St. Louis, Missouri.
ACID
BY FISHES
583
pmoles of p-nitrobenzoic acid, and 340 pmoles of potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Nitro reductase activity was determined at 540 rnp after trichloroacetic acid precipitation of protein, by diazotizing and coupling p-aminobenzoic acid to N-(l-naphthyl)ethylenediamine to yield a colored derivative as described by Fouts and Brodie (2). Appreciable quantities of p-aminobenzoic acid were also acetylated (2,s) by fish tissue preparations. The extent of conjugation was determined by heating aliquots of the trichloracetic acid supernatant in boiling water for 30 minutes (2, 8) and then diazotizing and coupling in the usual manner (2) to give the total (conjugated and free) p-aminobenzoic acid. Free paminobenzoic acid accounted for 30-35aj, of the total amine produced by liver 10,OOOgsupernatant and soluble fractions, and 45-50% by corresponding kidney fractions. There was no appreciable change in these ratios with pH, fish species, incubation temperature, cofactor and inhibitor concentration, or incubation time. The ratio of free to conjugated amine was relatively constant; consequently, the majority of incubation samples were only assayed for free p-aminobenzoic acid and these data are presented in this report. NADPH-cytochrome c reductase was assayed by following the changes in absorbancy at 550 rnr TABLE TISSUE
DISTRIBUTION
I
OF NITRO
ACTIVITY
REDUCTME
a
rmoles p-aminobenzoic acid fomedigm tissue/hour TiSSW
Liver Blood Muscle Anterior Posterior Heart Brain Spleen Testes
kidney kidney
Rainbow trout
Steelhead trout
Chinook salmon
0.230 0.002 Nil 0.023 0.120 Nil Nil 0.003 Nil
0.171
0.248
0.006 0.053
Nil
Nil 0.035 0.173 0.010 0.003 Nil
~1The incubation mixture contained 0.5 pmole NADP, 1.25 pmoles MgClz, 10 pmoles glucose g-phosphate, 1 Kornberg unit glucose 6-phosphate dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml 10,OOOg supernatant fraction from male fish (equivalent to 0.34 gm tissue), and 340 rmoles potassium phosphate buffer (pH 7.4), in a final volume of 5 ml. Incubations were carried out for 2 hours at 25” under nitrogen.
584
BUHLER TABLE
REQUIREMENTS ZOIC ACID
AND
II
FOR REDUCTION BY FISH LIVER
OF p-NITROBENPREPARATIONS~ ~moles -aminobenozic XL2 formed/gm liver/hour Rainbow Coho trout salmon
System
Complete Minus NADP Minus glucose 6-phosphate Minus Mgz+ Minus glucose B-phosphate dehydrogenase Plus air Minus all additives a Incubation conditions described in Table I.
0.168 0.032 0.102 0.164 0.158
0.191 0.07:)0.078 0.180 0.188
0.010
0.040 0.037
were the same as those
in a Beckman model DB spectrophotometer as described by Williams and Kamin (9). NADPHOxidase was determined spectrophotomet’rically at 340 rnp in the manner described by Gillette et al. (10). The glucose 6-phosphate dehydrogenase content of appropriately diluted fish liver preparations was measured by following the increase in absorbancy at 340 mp (11). Chemical assays. Lipid peroxidat,ion activit,y was measured by determining the amount of malonaldehyde formed with the thiobarbituric acid method of Ottolenghi (12). Lipid peroxide values have been expressed in terms of optical density at 530 rnp corrected for peroxide value due to the reagent blank.
RASMUSSON
activities were obtained on addition of NADP, glucose 6-phosphate, and a low concentration of MgClz (Table II). Enzyme activity was reduced appreciably by omission of NADP or glucose 6-phosphate and almost completely in an oxygen atmosphere. The rate of reduction of p-nitrobenzoic acid by trout supernatant fraction, 0.88 Imole of substrate reduced per gram liver per hour, was linear over the time interval examined (Fig. 1). Under aerobic conditions, however, t,he system rapidly lost activity. Some formation of p-aminobenzoic acid still occurred during the initial phases of the aerobic reaction, suggesting that the inhibition by oxygen was not an immediate process as one would expect if oxygen were acting as a competing electron acceptor, i.e., an NADPH-oxidase. Instead it appears that inhibition by oxygen occurred gradually, perhaps reflecting the slow oxidation of some sensitive component of the system. The effect of pH on nitro reductase activity is shown in Fig. 2. In phosphate buffer the optimum pH of the trout liver system was 7.4. The pH optima in pyrophosphate
RESULTS
Tissue distribution of enzyme activity. Incubation of p-nitrobenzoic acid with the 10,OOOgsupernatant7 fraction from various fish tissues showed that the liver and the posterior portion of the kidney were capable of reducing aromatic nitro groups (Table I). Anterior kidney and other fish tissues exhibited negligible activity. Previous studies in rabbits have shown that kidney tissue has one-half of the nitro reductase activity of liver (2). Requirements of the liver enzyme system. Requirements for the reduction of p-nitrobenzoic acid by fish liver supernatant fraction were similar to those reported for mammalian nitro reductase (2). Maximal 7 Homogenates pattern.
showed
essentially
the
same
0 0
1
2
3
4
Time IhoursI
FIG. 1. Reduction of p-nitrobenzoic acid by male rainbow trout liver supernatant fraction as a function of time. The incubation conditions were the same as those described in Table I and were carried out for the indicated time in air or nitrogen.
REIXJCTION
OF p-NITROBENZOIC
and Tris buffers were 8 and 7.6, respectively. Nitro reduct)ase activities in pyrophosphate or Tris buffers were about 70 and 30 % greater, respectively, than those observed with phosphate. .
ACID
,585
BY FISHES
Fish liver supernatant fraction which was heated for 2 minutes in boiling water and then cooled was completely inactivated. Frozen tissue samnles retained most of their activity, but subcellular preparations were partially inactivated by freezing or prolonged storage in the cold. The fish liver nitro reductase svstem was unusually sbable toward increases in temperature under the anerobic assay conditions (Fig. 3). Enzyme activity continued to increase with temnerature uruil 60” was reached. Above this temperature, the rate of reduction leveled off and then remained constant for incubation t,emperatures approaching 75”. Preincubation of the enzyme and cofactors at 25” in air in the absence of substrate, however, resulted in a substantial decrease in activity. When aerobic preincubation of fish liver enzyme was carried out for 1 hour at temneratures above 30”. enzyme activity was completely lost. There was no loss in nitro reductase activity if aerobic preincubation took place in 2 mM cysteine or in 10 rnM sodium pyrophosphate buffer, pH 7.4. Localization of enzyme activity. In contrast to the mammalian system (a), fish liver nitro reductase activity was not detected in microsomes but was confined to the 105,OOOg supernatant fraction (Table III). Significant microsomal activity could not be demonI
1
l,/
I
PH
FIG. 2. Relation of pH and nitro reductase activity with male rainbow trout supernatant preparation. Incubation conditions were the same as those described in Table I with 340 rrmoles of potassium phosphate, sodium pyrophosphate, or Tris buffer added as indicated.
2 c
5 .E 2
TABLE 0.300
INTRACELLULAR t
III
L~C.~LIZ.~TI~N
REDUCTMR
Intracellular fraction
-
pm&s p-aminobenzoicacid formed/gm liver/hour *T:t
01 0
I 20
I 40
I MI
I 80
Temperature hi
FIG. 3. Effect of incubation temperature on reduction of p-nitrobenzoic acid by male rainbow trout liver supernatant preparation. The incubation condit,ions were the same as those described in Table I except that incubations were carried out for 1 hour at the indicated temperatures.
Whole homogenate 10,000 gsupernatant fraction Microsomes Soluble fraction (103,OOOg supernatant fraction) Microsomes + trace solubleb
OF Nrrriro
ACTIVITY~
Coho salmon
Rainbow trout
0.444
0.166 0.159
0.190 0.186
0.021 0.326
0.004 0.161
0.008 0.145
0.057
0.032
0.024
y The incubation conditions were the same on those described in Table I. * Soluble fraction (0.1 ml) was added t.o 1 ml of the microsomal preparation.
586
BUHLER
AND
strated even with addition of a small quantity of the soluble fraction to the assay system. An appreciable sedimentation of nitro reductase activity was achieved by prolonged centrifugation of fish liver soluble fraction at 105,OOOgfor 8 hours (Table IV). Maximum activity was associated with a zone which also contained a flocculent reddish precipitate. An ultrafiltrate of fish liver soluble preparation was essentially devoid of nitro reductase activity (Table V), thus confirming the enzymic nature of the fish liver system. Enzyme activity was not lost by this treatment but instead remained concentrated in the residue from the ultrafiltration. Overnight dialysis of the soluble fraction against isotonic KC1 at 4” resulted in a significant loss of nitro reductase activity (Table V), while activity was retained in undialyzed material stored overnight under comparable conditions. Similar results were observed with the 10,OOOg supernatant fraction. The dialyzed enzyme could not be reactivated by addition of NADPH, flavin, or other cofactors. Addition of the ultraTABLE
IV
SEDIMENTATION OF FISH LIVER NITRO REDUCTASE ACTIVITY~ Fraction
Soluble fraction natant fraction) Upper aoneb Middle zoneb Bottom zoneb
(105,OOOg super-
rmoles p-aminobenozic acid formed/pm liver/hour 0.170 0.018 0.058 0.380
a The incubation conditions were the same as those described in Table I except that 1 ml of male coho salmon liver soluble fraction was employed. b The soluble (105,OOOg supernatant fraction) was centrifuged at 105,OOOg for 8 hours. The contents of the centrifuge tube were separated into three zones: upper (one-fourth of tube), containing a white flocculant suspension; middle (one-half of tube), clear yellowish zone; and bottom (one-fourth of tube), containing a reddish precipitate.
RASMUSSON TABLE V EFFECTS OF DIALYSIS AND ULTRAFILTRATION NITRO REDUCTASE ACTIVITJP Fraction
Soluble (105,OOOg supernatant)” Ultrafiltrate from solublec Residue from ultrafiltrationd Dialyzed soluble” Dialyzed soluble + ultrafiltrate from solublef Dialyzed soluble + cysteine (2 mM)
ON
fimoles of p-aminobenzoic acid formed’gm liver/hour 0.112 0.009 0.102 0.022 0.028 0.162
a The incubation conditions were the same as those described in Table I. This experiment used enzyme from male rainbow trout. b Stored overnight at 4”. c Prepared with a LKB Ultrafilter. d Reconstituted to the original volume with 0.154 M KCI. e Dialyzed overnight at 4” against 0.154 M KCl. ’ One ml of each preparation added to the incubation mixture.
filtrate from the soluble fraction also had little effect on the activity of the dialyzed preparation. Nitro reductase activity, however, could be restored to the original level or even stimulated by inclusion of cysteine in the incubation media or in the dialysis mixture. The reactivation by cysteine suggests that the loss of activity during the dialysis was probably an oxidative process. Dialysis apparently removed some low molecular weight components of the soluble fraction which normally protects the enzyme system against oxidative inactivation, and cysteine was able to substitute for these missing factors. Dialyzable factors in the soluble fraction have been reported to prevent the oxygen-induced respiratory decline of rat microsomes or mitochondria (13). Requirement for NADPH. NGtro reductase activity in the fish liver system was reduced significantly in the absence of added NADP or glucose 6-phosphate (Table II), indicating that the fish system required a reduced form of the coenzyme. The optimum concentration for added NADP was about 0.3 rmole (0.06 mM) (Fig. 4). Substitution of chemically reduced NADPH for the NADPHgenerating system gave a 30% stimulation
REDUCTION
OF p-NITROBENZOIC
in activity (Table VI), which is consistent with the requirement for reduced pyridine nucleotide. The isocitric dehydrogenase NADPH-generating system was somewhat more effective than the glucose g-phosphate dehydrogenase system, presumably because of the presence of glucose 6-phosphatase in the fish tissue preparations. Substitution of preformed NADH or a NADH-generating system was less effective than NADPH. Species diferences in enzyme activity. Table VII compares nitro reductase activity in liver preparations from various freshwater and anadromous fish species. No appreciable differences in enzyme activity as related to sex were noted in the limited number of samples tested; consequently the results were averaged without regard to the sex of the fish. Reduction of p-nitrobenzoic acid was greatest in preparations from the American shad and coarsescale sucker and least with the Pacific lamprey. Liver nitro reductase levels were comparable in juvenile and spawning adult silver salmon. The activities of liver preparations from most fish species were similar to that of the rat liver system assayed under the same conditions (Table VIII). Chan and co-workers (14) have reported a synergistic interaction of combined rat and trout liver preparations on cyclodiene
q,,
, 0
2 NADP Concentration
, 4
,
( 6
x 10m4M
FIG. 4. Effect of NADP concentration on the reduction of p-nitrobenzoic acid by male rainbow trout 10,OOOgsupernatant fraction. The incubation conditions were the same as those described in Table I except that the indicated amount of NADP was employed.
ACID
BY FISHES TABLE
REQUIREMENT
587 VI
FOR NADPHa
NADP (0.5 rmole), glucose B-phosphate (10 moles), and 1 unit glucose 6-phosphate dehydrogenase NADP (0.5 rmole), sodium isocitrate (10 pmoles), and 2 mg isocitric dehydrogenase NADPH (1 pmole) NADPH (3 pmoles) NADH (3 pmoles) NAD (0.5 pmole) and nn-malate (10 pmoles)
0.195
0.248
0.276 0.320 0.222 0.148
n The incubation mixture contained 1.25 pmoles MgC12, 3 pmoles p-nitrobenzoic acid, 1 ml male rainbow trout liver 10,OOOg supernatant fraction, and 340 pmoles potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Incubations were for 2 hours at 25” under nitrogen. b The mean of three such experiments are presented.
epoxidation. Incubation of fish and rat liver supernatant fractions together, however, gave a combined nitro reductase activity that was only a summation of the two individual activities (Table VIII). Total nitro reductase activity was more than doubled, however, when catfish and rainbow trout fractions were incubated together. Thus it appears that certain species of fish may exhibit a low nitro reductase activity because of a deficiency in some component of the enzyme system. This missing factor was apparently absent from rat liver preparations but could be supplied by the liver supernatant fraction from another fish species. The synergistic interaction of mixed fish preparations was largely eliminated when incubations were carried out in sodium pyrophosphate buffer (Table VIII), suggesting that the unknown fish factor and pyrophosphate have similar effects. The unknown component in fish preparations which stimulates the fish nitro reductase system may be related to the dialyzable protective agent in the soluble fraction previously described. Flavin requirements of the nitro reductase
585
BUHLER
AND
system. Mammalian liver nitro reductase has been shown to be a flavoenzyme by Fouts and Brodie (2), and Adamson and coworkers (6) have demonstrated the stimulatitin of shark liver nitro reductase by added flavin. The teleost liver nitro reductase system was also greatly stimulated by added flavin (Table IX). Flavin adenine dinucleotide (FAD), flavin mononucleotide TABLE RBDUCTION
VII
OF P-NITROBKNZOIC ACID VARIOUS FISH SPECIIB~
OF
pmoles of p-aminobenzoic acid formed.’ anl”yK
Fish speck&
Smallmouih bass (2) Bluegill (2) Carp (4) Channel catfish (4) Flathead catfish (2) White crappie (1) Pacific lamprey (8) Yellow perchc Northern pike (1) Chinook salmon (11) Coho salmon (14) Coho salmon fingerlings” Sockeye salmon (8) American shad (10) White sturgeon (8) Largesca!e sucker (7) Rainbow trout (20) SteeIliead trout (8)
IN LIVI~R
0.178 0.091 0.331 0.062 0.050
(2)
+ 0.006 f 0.009 f 0.053 f 0.009 f 0.017 0.072 0.019 f 0.003 0.072 f 0.019 0.143 0.253 f 0.037 0.177 f 0.043 0.228 f 0.018 0.312 f 0.092 0.485 f 0.098 0.082 f 0.027 0.559 & 0.073 0.198 f 0.039 0.183 f 0.044
a Metabolism expressed as mean pmoles of paminobenzoic acid formed per gram liver per hour f standard deviation), (each preparation was assayed in duplicate), averaged without regard to sex. The incubation mixtures contained 0.3 pmole NADP, 1.25 pmoles MgClz, 10 pmoles glucose g-phosphate, 1 Kornberg unit glucose 6phosphat’e dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml of liver 10,OOOgrupernatant fraction (equivalent to 0.34 gm liver), and 340 pmoles potassium phosphate buffer (pH 7.4) in a total volume of 5 ml. Incubations were for 2 hours at 25” under nitrogen; b Number of adult, animals per mean is ex-
pressed in parentheses. c Livers of several fish were pooled to obtain sufficient enzyme fo? assay. Mean of two such pools of adult yellow perch and fingerling silver salmon reported. Fingerling silver salmon averaged 20 gm per fish.
RASMUSSON TABLE NITRO
REDUCTUE SND RAT
VIII
ACTIVITY OF COMBINED ENZYME PREPARATIONS~
FISH
rmoles p-aminobenwic acid formed/gm liver/hour Fish enzyme
None Coho salmon 10,OOOgsupernat,ant fraction Rainbow trout 10,OOOg supernatant fraction Channel catfish 10,OOOg supernatant fraction Rainbow trout + Channel catfishsupernatant frac-1 tion
0.178
0.148” 0.285
0.160’
0.300”
0.030
0.3i4 0.128 0.585
a The incubation mixture contained 0.5 rmole NADP, 1.25 pmoles MgC12, 10 Fmoles sodium isocitrate, 2 mg isocitric dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml of fish liver supernatant fraction (equivalent to 0.34 gm liver), and 340 rmoles potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Incubations were carried out for 2 hours at 25’ under nitrogen. b Contained fish enzyme plus 1 ml of 0.154 M KC1 except in the mixed rainbow trout and channel catfish experiment. c Prepared from adult male Sprague-Dawley rats. One milliliter of supernatant preparation was added to the incubation mixture. d One milliliter of 0.05 M sodium pyrophosphate buffer (pH 7.4) was added to each incubation flask. e The average of two separate experiments.
(FMN), and riboflavin all enhanced activity, but FAD appeared to be most effective. Soluble and dialyzed soluble preparations also responded to flavins, albeit the magnitude of effect was considerably less. Soluble fraction was subjected to mild acid treatment to dissociate the flavin prosthetic group (15), and nitro reductase activity of the acid treated preparation was partially restored by added flavin. Acid treatment of the enzyme, however, caused an appreciable loss in activity. A similar susceptibility to denaturation upon mild acid treatment was reported for the azo reductance system from mammalian liver (16). In contrast, the activity of mammalain nits reductase was
REDUCTION TABLE EFFECT
OF p-NITROBENZOIC
IX
OF ADDED FLAVIN ON RAINBOW NITRO REDUCTASEO
TROUT
pm&s @minobenzoic acid formed/gm liver/hour Additive
None 0.01 mM FAD 0.1 mM FAD 1 mM FAD 0.01 mM FMN 0.1 mM FMN 1 mM FMN 0.01 mM Riboflavin 0.1 mM Riboflavin 0.7 mM Riboflavin
0.274 0.356 1.07 1.55 0.410 0.868 1.41
0.010
0.200
0.051
Nil
0.032 0.109
0.542 0.650
0.150
0.023 0.042
0.514 0.616
0.136
0.088
0.023 0.059
o.2601 I I I 0.405 1.32
(LThe incubation conditions were the same as those described in Table I. There was no detectable reduction of p-nitrobenzoic acid by an NADPH-generating system with added flavin in the absence of enzyme. b Soluble fraction was 105,OOOg supernatant fraction. c Dialyzed overnight at 4” against 0.154 M KCl. d Treated according to Zelitch and Ochoa (15).
ACID
Activation of fish nitro reductase by SH-type compounds is reminiscent of the bacterial nitro reductase system described by Saz et al. (17). The Escherichia coli required enzyme was NAD-dependent, cysteine or glutathione and Mn2+ for its activation, and was inhibited by low concentration of chlortetracycline. The fish and bacterial nitro reductase systems are not identical since the fish system was not inhibited significantly by chlortetracycline, nor was Mn2+ required for its activation. Moreover, EDTA stimulated the fish system but produced an inhibition of the bacterial enzyme, and the bacterial system retained appreciable activity under aerobic conditions while the fish enzyme did not,. Nicotinamide stimulated the mammalian system (a), but at a concentration of 20 rnn1 it caused a 40% inhibition of the fish liver nitro system (Table XI). reductase Nicotinamide also inhibited nitro reductase activity in washed fish liver microsomes and soluble and dialyzed soluble fractions. High concentrations of magnesium ions also produced a significant inhibition of the fish system in a manner analogous to that reported for mammalian enzyme (2). A slight stimulation in nitro reductase activity TABLE
not lost by such acid treatment, and added flavin completely reactivated the enzyme (2). Although washed fish liver microsomes produced only negligible reduction of p-nitrobenzoic acid, modest increases in activity were observed following incubation in the presence of high concentrations of flavin. E$ect of additives. In addition to flavin, a variety of other compounds stimulated the reduction of p-nitrobenzoic acid by fish liver preparations. The chelating agents EDTA, 8-hydroxyquinoline, and pyrophosphate increased nitro reductase activity, while chlortetracycline and cr,ar-dipyridyl were without effect (Table X). Nitro reductase activity was enhanced by addition of glutathione, 2-mercaptoethanol, or cysteine, the latter being most effective. Such sulfhydryl compounds were without affect on the mammalian enzyme (2).
589
BY FISHES
EFFECT
X
OF VARIOUS CHELATING AGENTS SULFHYDRYL COMPOUNDS ON VITRO REDUCTASE ACTIVITY~ Additive
Chlortetracycline . HCl LY,a-Dipyridyl Ethylenediaminetetraacetic acid (EDTA) 8-Hydroxyquinoline Sodium pyrophosphate, pH 7.4b Cysteine Glutathione Z-Mercaptoethanol
Concenn~~ion
AND
% S$n+3tion or mhlbltion
0.2 0.5 1
75 -9 +56
0.1 10
+114
2 2 2
+61 $29 $36
+21
a The incubation conditions were the same as those described in Table VIII. The assay mixture contained 1 ml of male rainbow trout liver 10,OOOg supernatant fraction (equivalent to 0.34 gm liver). b Potassium phosphate buffer was omitted from the incubation medium.
590
BUHLER TABLE
AND
XI
EFFECT OF VARIOUS INHIBITORS ON NITRO REDUCTASE ACTIVITY” Additive
M&L M8.L MgClz MgClz Nicotinamide Nicotinamide Nicotinamide Nicotinamide p-Chloromercuribenzoate p-Chloromercuribenzoate NaCN NaCN NaNB NaNs Carbon monoxide-nitrogenb Carbon monoxide (IOOY,) SKF-525A None, preincubated in air 15 min SKF-525A, preincubat,ed in air 15 min
Concentra-% ;;:i:tion (mx)
inhibition
0.25 1 5 15 1 5
+13 -7 -23 -34 -5
10
-25 -40 -22
20
0.1 1 0.1 1 0.1 1
1
-18
-80 -73 -91 -38 -36 -20 -40
-8 -55
1
-58
a The in:ubation conditions were the same as those described in Table VIII except that the indicated concentration of MgCl, was employed. The assay mixture contained 1 ml of male rainbow trout liver 10,OOOg supernatant fraction (equivalent, t,o 0.34 gm liver). b Carbon monoxide-nitrogen (1:4).
was observed at a magnesium concentration of 0.25 mM; therefore this level was selected for normal incubation conditions. The sulfhydryl binding agent p-chloromercuribenzoate (PCMB) almost completely inhibited the fish nitro reductase system (Table XI). Sodium cyanide and sodium azide also caused a significant inhibition of the system. Carbon monoxide reduced fish liver nitro reductase activity by 40%, suggesting the involvement of a carbon monoxide-sensitive hemoprotein component such as cytochrome P-450 (5). The microsomal oxidase inhibitor SKF-525A had little effect on nitro reductase activity. Although aerobic preincubation of the system with SKF-525A in the absence of substrate (18) did cause a sharp drop in activity, a similar loss occurred upon preincubation of the enzyme in the absence of
RASMUSSON
added SKF-525A. Decreased activity, therefore, must be ascribed to the oxygen-derived inactivation of the enzyme system as previously described. In addition, metal ions (Cuz+, Caz+, Mnz+, and Co2+) were without effect on nitro reductase activity at concentrations of 0.1 and 1 mM, while 0.1 mM Fe2+ caused a slight increase in activity. Lipid peroxidation. As measured by the thiobarbituric acid reaction, an appreciable peroxidation of lipids occurred in fish liver preparations (Table XII) in a manner similar to that previously reported for mammalian tissue (19, 20). Formation of lipid peroxide was greatest with washed microsomes and least in the soluble fraction.8 Addition of a trace of the soluble fraction to microsomes caused a significant reduction in the formation of thiobarbituric acid-reacting substances. A similar depression in microsomal peroxide formation by the soluble fraction has been reported for rat preparations (13). Lipid peroxide apparently accumulated during preparation of fish liver subcellular fractions since appreciable thiobarbituric acid-reacting material was detected at the start of the incubation period (Table XII). It is interesting to note that whereas this initial value increased with subsequent incubation in air, thiobarbituric acid color actually decreased following anerobic incubation. These results suggest that in the absence of oxygen some component of the incubation mixture was capable of reducing lipid peroxides or their decomposition products. Addition of pyrophosphate buffer to the incubation mixture caused a marked reduction in the thiobarbituric acid-reacting material derived from fish liver subcellular of fractions, and no further formation peroxides occurred when incubations were carried out in the presence of pyrophosphate or cysteine. Pyrophosphate has been previously found to inhibit lipid peroxidation in rat liver microsomes (21). Fish liver 10,OOOgsupernatant preparations contained five to six times as much glucose 6-phosphate dehydrogenase as did corresponding rat preparations. However, * The protein content of these fractions was not, determined, and lipid peroxidation might be more :omparable on a per milligram protein basis.
REDUCTION
OF p-NITROBENZOIC TABLE
INFLUENCE
OF VARIOUS AGENTS ON LIPID
ACID
XII
PEROXIDE FORMATION BY FISH LIVER
10,OOOg supernatant fraction 10,OOOg supernatant fraction 10,OOOg supernatant fraction 10,OGOg supernatant fraction Microsomes Soluble (105,OOOg supernatant fraction) Microsomes + trace solubleb
tion time (hours)
Air None
NaPPi
Cyst&e
NOW
0 0.5 1
0.122 0.174 0.167
0.069
0.112
0.062
0.122
0.122 0.115 0.101
2
0.176 0.550
1 1 1
PREPARATIONS?
ODrn
IDCUt%+
Fraction
591
BY FISHES
Nitrogen NaPPi
Cysteine
0.069
0.112
0.063
0.106
0.075 0.058
0.293
0.039 0.360
QThe incubation conditions were the same&sthose described in Table VIII. The reaction mixture contained 1 ml female rainbow trout liver preparation (equivalent to 0.34 gm liver). As indicated, 340 pmoles sodium pyrophosphate buffer (pH 7.4) (added in place of potassium phosphate buffer) or 10 pmoles cysteine were added to the incubation system. Incubations were carried out at 25” for the indicated time under either air or nitrogen. At the end of the incubation period, the reaction mixture was assayedfor lipid peroxide by the method of Ottolenghi (12). b Soluble fraction (0.1 ml) was added to 1 ml of the microsomal preparation. no significant correlation between fish liver glucose &phosphate dehydrogenase levels and nitro reductase activity was observed. Kamm and Gillette (4) have suggested that anerobic reduction of nitro compounds and aerobic oxidation of NADPH may be catalyzed by the same enzyme system (presumably NADPH-cytochrome c reductase), but no direct relationship between NADPH-oxidase and nitro reductase levels could be found in fish liver preparations. Fish liver microsomes contained little nitro reductase activity, although the NADPHcytochrome c reductase levels of microsomes was about eight times greater than that of the soluble fraction.9 A similar concentration of NADPH-cytochrome c reductase has been reported in liver microsomes from the rat (22) and rainbow trout (14). Overnight dialysis of the fish liver soluble fraction against 0.154 M KC1 resulted in a complete loss of endogenous cytochrome c reduction, but the level of NADPH-cytochrome c reductase remained unchanged. Dialysis of g The soluble fraction catalyzed an appreciable reduction of cytochrome c in the absence of added NADPH. In such cases, NADPH-cytochrome e reductase was determined by measuring the difference in the rates of reduction of cytochrome c in the presence and absence of added NADPH (22). The mean activity of rainbow trout liver microsomes was 7.2 units/gm liver, and that of soluble fraction was 0.9 unit/gm liver.
the soluble fraction had been found to produce an appreciable loss of nitro reductase activity (Table V). Effect of diet. No significant changes in liver 10,OOOg supernatant nitro reductase activity were found when groups of rainbow trout were starved for 1, 2, 4, and 8 weeks. These results are to be contrasted with those of Mato and Gillette (23), who observed significant increases in the nitro reductase activity of rat liver microsomes induced by starvation. Feeding rainbow trout diets high in protein or carbohydrate also failed to cause significant changes in liver nitro reductase levels. Oral administration or intraperitoneal injection of various inducing agents (phenobarbital, phenylbutazone, DDT, and chlordane) failed to produce a consistent increase in fish liver nitro reductase activity.lO lo Yearling rainbow trout were fed diets containing 10 or 25 mg technical or p,p’-DDT/lOO gm diet, and groups of fish were assayed at I, 2, and 4 weeks; were given daily intraperitoneal injections of corn oil containing 50 mg/kg technical DDT or chlordane for 4 days and assayed on the fifth day; or were given daily oral administration of 50 mg/ kg phenobarbital or phenylbutazone for 4 days and assayed.on.the fifth day. Fingerling coho salmon were given daily intraperitoneal injections of corn oil containing 50 mg/kg chlordane for 4 days and assayed on the fifth day.
BUHLER
AND
RASMUSSON
12
8 % x -I>
I/O -1
1 0
I
I
I
2
I
I
4
I 6
II rs1 x lo4 0
2.0
1.0
3.0
ml. Enzyme
FIG. 5. Effect of added enzyme on reduction of p-nitrobenzoic acid by male rainbow trout liver supernataht fraction. The incubation conditions were the same as those described in Table VIII with the addition of the indicated amount of male trout liver 10,OOOg supernatant fraction. Control experiment carried out in duplicate. Third experiment with 2 mM cysteine added to above incubation mixture.
Reaction kinetics. A plot of nitro reductase activity versus amount of rainbow trout liver supernatant enzymel’ was nonlinear at low enzyme concentrations (Fig. 5). Enzyme activity can be reduced in this characteristic manner at low enzyme levels if the enzyme preparation or incubation mixture contains a limited amount of a dissociable activator or coenzyme, or if an inhibitor of the system which could be gradually destroyed is present (24). The shape of the trout enzyme activity curve was not altered by omission of Mg2+ from t.he incubation medium. Substitution of NADPH for the NADPH-generating system or additions of lO- 4 M FNM failed to restore linearity, although the slope increased I1 Chinook and coho salmon 10,OOOgsupernatant preparations gave comparable results. Crude rainbow trout liver homogenates and soluble fraction behaved similarly.
FIG. 6. Double reciprocal plot of p-nitrobenzoic acid concentration against rate of reduction by male rainbow trout liver supernatant fraction. Velocities are given as moles/liter/hour, and substrate concentrations are in moles/liter. The incubation conditions were the same as those described in Table VII with the addition of the indicated amounts of p-nitrobenzoic acid to a total volume of 6 ml.
appreciably. Nitro reductase activity became directly proportional to enzyme concentration, however, when 2 lll~ cysteine was included in the incubation mixture (Fig. 5). As shown in Fig. 6, from the LineweaverBurk plot, the apparent K, value for the reduction of p-nitrobenzoic acid by rainbow trout supernatant fraction was about 1.03 X W4 M. Although the Michaelis constant for the mammalian nitro reductase system has not been reported, the Km for the trout liver system was about the same as those reported for the oxidation of various drugs by rat liver microsomes (25). Products of the reaction. An aliquot of the trichloroacetic acid filtrate from a typical incubation mixture was subjected to thinlayer chromatography on a silica gel GFl2 developed in butanol saturated with water. A single amine-containing compound with a mobility identical to that of authentic I2 Brinkman York.
Instrument
Co., Great Neck,
New
REDUCTION
OF p-NITROBENZOIC
p-aminobenzoic acid was detected after spraying the dried plate with diazotized sulfanilic acid. DISCUSSION
This investigation has shown that teleost fishes are capable of reducing p-nitrobenzoic acid by an NADPH-requiring enzyme system located primarily in the liver. Previous reports have indicated that the nitro reduct,ase system in mammalian liver is distributed between the microsomal and soluble portions of the cell (2). The fish enzyme, in contrast, was located predomiand nately in the soluble fraction, microsomes contained little activity. It is possible that the fish enzyme is actually localized in the endoplasmic reticulum in the intact cell but that it is more easily dissociated from the microsomes during isolation than is the mammalian system. The fish nitro reductase system, however, could be sedimented along with a very light particulate fraction when fish liver “soluble” enzyme was subjected to further centrifugation, suggesting that the fish nitro reductase system was associated with very light microsomes as previously described for the soluble mammalian enzyme (26). Mammalian azo reductase, which is similar in many respects to the nitro reductase system, is also found primarily in the soluble portion of the cell (16). The fish liver nitro reductase system was strongly inhibited by oxygen. This inhibition presumably reflected oxidation of some essential component of the system since liver preparations which were preincubated in air were no longer active. Some aerobic inactivation of the fish liver system apparent,ly occurred during preparation of the subcellular fract’ions and during the short period required for nitrogen to flush air completely from the incubator-shaker. Mammalian Intro reductase is also inhibited by oxygen (2), and recent studies have shown that preincubation of rat liver microsomes for 60 minutes under oxygen complet’ely destroyed nitro reductase activity while the system was completely stable under nitrogen (27). The oxygen derived inactivation of the fish nitro reduct,ase system may be related
ACID
BY FISHES
593
to the peroxidation of liver lipids. Mammalian liver preparations are known to undergo spontaneous lipid peroxidation (19, 20), and addition of hematin compounds (281, ascorbic acid (12), or heavy metals (12, 29) stimulates lipid peroxide formation by subcellular fractions. An NADPH-linked peroxidation of lipids in rat liver microsomes has also been reported (30, 31). Such lipid peroxides have a deleterious effect on many proteins and enzymes (19, 32, 33). Enzymes containing essential sulfhydryl groups appear to be especially susceptible to inactivation by lipid peroxides (33). Peroxidation of unsaturated fatty acids in the phospholipid containing microsomal membranes may be responsible for the inactivation of certain mammalian liver microsomal mixedfunction oxidases during aerobic incubation (34) * Antioxidants such as cysteine (33) and a-tocopherol (21, ZS), and chelating agents like EDTA and pyrophosphate (21)) prevent peroxidation of lipids in mammalian liver preparations. Cysteine also forestalled inactivation of the rat liver microsomal enzyme which hydroxylates napthalene (35). Other factors may be important, however, since ar-tocopherol failed to maintain the microsomal enzymes which metabolizes hexobarbital and codeine even though lipid peroxidation was abolished (34). Rainbow trout liver nitro reductase that had been inactivated by oxygen was restored to complete activity by addition of cysteine or sodium pyrophosphate. The trout enzyme was normally protected against oxygen inactivation by unknown dialyzable components of the soluble fraction (Table V). The relative instability of washed mammalian liver microsomal preparations is thought to be due t#o removal of such stabilizing factors (36). At low enzyme concentrations the fish liver nitro reductase system may be more susceptible to oxidative inact,ivation, as evidenced by the nonlinearity of the enzyme concentration-yield 1curve. Under these conditions limited amount,s of antioxidants in the soluble fraction apparently afford insufficient protection. Added cysteine, however, prevents oxygen damage and restores linearity to the enzyme concentration-yield curve (Fig. -5). The effectiveness
594
BUHLER
AND
of PCMB as an inhibitor of fish liver nitro reductase suggests that some component of the system contains essential sulfhydryl groups. One may speculate, therefore, that an initial peroxidation of lipids in fish liver preparations is followed by an inactivation of the nitro reductase system through oxidation of sensitive sulfhydryl groups (33). Cysteine and similar compounds probably serve as antioxidants and thus protect the system. Pyrophosphate may prevent inactivation of the nitro reductase system by chelating heavy metals which catalyze lipid peroxidation (12, 29), or it may stimulate the system by binding inhibiting magnesium ions (2) or maintaining NADPH levels through inhibition of endogenous pyrophosphatases (37). Nicotinamide is routinely added to the incubation mixture in most drug enzyme studies since it has been shown to stimulate reductase and mammalian liver nitro mixed-function oxidases (2, 38). Nicotinamide is thought to enhance drug metabolism in vitro by preventing the destruction of NADP by NADase (39). In fishes, however, nicotinamide has now been shown to be a good inhibitor of the liver nitro reductase system.13 In this connection, Schenkman et al. (40) have recently reported that nicotinamide does inhibit mammalian liver microsomal NADPH-dependent, mixedfunction oxidase, probably by interaction with cytochrome P-450. There is good evidence that the mammalian nitro reductase system contains cy$ochrome P-450 (5) and NADPH-cytochrome c reductase activity (4). The sensitivity of fish liver nitro reductase toward carbon monoxide (5) and nicotinamide (40) suggests that cytochrome P-450 may also be a constituent of the fish enzyme. In this connection, the cytochrome P-450 content of fish liver is known to be quite high (14, 41). Although NADPH-cytochrome c reductase may be involved in the mammalian nitro reductase system (4), fish liver micro13Nicotinamide also inhibits the liver microsomal mixed-function oxidase from fishes (D. R. Buhler and M. E. Rasmusson, Comp. Biochem. Physiol., in press).
RASMUSSON
somes which contain the majority of the NADPH-cytochrome c reductase are devoid of nitro reductase activity. In addition, the nitro reductase activity of the fish soluble fraction is greatly reduced by dialysis, whereas the NADPH-cytochrome c reductase activity remains unchanged. It seems apparent, therefore, that if NADPHcytoehrome G reductase is involved in the fish nitro reductase system, it is not the oxygen-sensitive or the rate-limiting component of the system. ACKNOWLEDGMENTS The authors acknowledge the diligent technical assistance of Miss Susan E. Anderson and Mrs. Donna R. Fink. We also thank Dr. Waldo S. Zaugg of the Western Fish Nutrition Laboratory, Cook, Washington, for his many helpful suggestions. REFERENCES 1. WESTFALL, B. B., J. Pharmacot. Exptl. Tbrup. 78, 386 (1943). 2. FOTJTS, J. R., AND BRODIE, B. B., J. Pharmacol. Exptl. Therap. 119,197 (1957). 3. MUELLER, W. A., 2. Physiol. Chem. 311, 155 (1958). 4. KAMM, J. J., AND GILLETTE, J. R., Life Sci., 2, 254 (1963). 5. GILLETTE, J. R., AND SASAME, H. A., Federation Proc. 24, 152 (1965). 6. ADAMSON, R. H., DIXON, R. L., FRANCIS, F. L., AND RALL, D. P., Proc. Natl. Acad. Sci. U.S. 64, 1386 (1965). 7. HITCHCOCK, M., AND MURPHY, S. D., Federation Proc. 26, 687 (1966). 8. HIETBRINK, B. E. AND DUBOIS, K. P., Radiation Res. 22, 598 (1964). 9. WILLIAMS, C. H., AND KAMIN, H., J. Biol. Chem. 237, 587 (1962). IO. GILLETTE, J. R., BRODIE, B. B., AND LADu, B. N., J. Pharmacol. Exptl. Therap. 119, 532 (1957). 11. KORNBERG, A., AND HORECKER, B. L., Methods Enzymol. 1, 323 (1957). 12. OTTOLENGHI, A., Arch. Biochem. Biophys. 79, 355 (1959). 13. GROVE, J. A., JOHNSON, R. M., AND CLINE, J. H., J. Biol. Chem. 241, 5564 (1966). 14. CHAN, T. M., GILLETT, J. W., AND TERRIERE, L. C., Comp. Biochem. Physiol. 20, 731 (1967). 15. ZELITCH, I., AND OCHOA, S., J. Biol. Chem. 201, 707 (1953). 16. FOUTS, J. R., KAMM, J. J., AND BRODIE, B. B., J. Pharmacol. Exptl. Therap. 120,291 (1957).
REDUCTION
OF p-NITROBENZOIC
17. SAZ, A. K., AND SLIE, R. B., Arch. Biochem. Biophys. 61,5 (1954). 18. GILLETTE, J. R., AND SESAME, H. A., Federation Proc. 23,537 (1964). 19. TAPPEL, A. L., AND ZALKIN, H., Arch. Biochem. Biophys. 80, 326 (1959). 20. Z~LKIN, H., AND TAPPEL, A. L., Arch. Biochem. Biophys. 88, 113 (1960). 21. CHATTERJEE, I. B., AND MCKEE, R. W., Arch. Biochem. Biophys. 110, 254 (1965). 22. PHILLIPS, A. H., AND LANGDON, R. G., J. Biol. Chem. 237, 2652 (1962). 23. KATO, R.,AND GILLETTE, J. R,. J. Pharmacol. Exptl. Therap. 160,279 (1965). 24. DIXON, M., AND WEBB, E. C. (eds.), “Enzymes,” Academic Press, New York (1964). 25. RUBIN, A., TEPHLY, T. R., AND MANNERING, G. J., Biochem. Pharmucol. 13, 1007 (1964). 26. FOUTS, J. R., Biochem. Biophys. Res. Commun. 6, 373 (1961). 27. GRAM, T. E., AND FOUTS, J. R., J. Pharmacol. Exptl. Therap. 162,363 (1966). 28. TAPPEL, A. L., AND ZALKIN, H., Nature, 186, 35 (1960). 29. MCKNIGHT, R. C., AND HUNTER, F. E., Biochim. Biophys. Acta 98, 643 (1965).
ACID
BY FISHES
595
30. HOCHSTEIN, P., AND ERNSTER, L., Biochem. Biophys. Res. Commun. 12, 388 (1963). 31. MAY, H. E., POYER, J. L., AND MCCAY, P. B., Biochem. Biophys. Res. Commun. 19, 166 (1965). 32. ROUBAL, W. T.,AND’TAPPEL, A.L., Arch. Biothem. Biophys. 113, 5 (1966). 33. WILLS, E. D., Biochem. Pharmacol. 7, 7 (1961). 34. GRAM, T. E., AND FOUTS, J. R., Arch. Biochem. Biophys. 114, 331 (1966). 35. BOOTH, J. AND BOYLAND, E., Biochem. J. 70, 681 (1958). 36. LEADBEATER, L., AND DAVIES, D. R., Biochem. PhurmacoZ. 13, 1607 (1964). 37. GILLETTE, J. R., GRIEB, W., AND SESAME, H., Federation Proc. 22, 366 (1963). 38. MITOMA, C., POSNER, H. S., REITZ, H. C., AND UDENFRIEND, S., Arch. Biochem. Biophys. 61, 431 (1956). 39. MANN, P. J. G., AND QUASTEL, J. H., Biochem. J. 36, 502 (1941). 40. SCHENKMAN, J. B., BALL, J. A. AND ESTABROOK, R. W., Biochem. Phurmacol. 16, 1071 (1967). 41. BRODIE, B. B., AND GILLETTE, J. R., American Chemical Society, Winter Meetings, 1966.
OF
BIOCHEMISTRY
AND
Reduction
BIOPHYSICS
103,
and Wildlife, Received
(1968)
of p-Nitrobenzoic
DONALD R. BUHLER2 Bureau of Sport Fisheries
.%?-,%j
May
AND
Fish-Pesticide
Acid
by Fishes’
MARY E. RASMUSSONa Research Laboratory,
25, 1967; accepted
September
Columbia,
Missouri
66.%‘01
7, 1967
A system which catalyzes the reduction of p-nitrobenzoic acid to p-aminobenzoic acid has been found in a number of freshwater fishes. The nitro reductase system occurred primarily in the liver, where it was associated with the soluble fraction of the cell. Fish liver nitro reductase required NADPH and was inhibited by oxygen. Preincubation of the system in air caused a loss of nitro reductase activity, suggesting that some sensitive component of the system had undergone oxidation. Some evidence was advanced relating this inactivation with the formation of lipid peroxides. Various sulfhydryl compounds and chelating agents could reverse or prevent the oxygen-derived inactivation. Additional evidence indicated that the fish liver nitro reductase system contained essential sulfhydryl groups and a carbon monoxide-sensitive component.
Aromatic nitro compounds are reduced under anerobic conditions to the corresponding amines by mammalian tissue homogenates (l-3). The enzyme responsible for these reductions occurs primarily in the liver in both the soluble fraction of the cells and in microsomes (2). Both NADPH and NADH can serve as electron donors for the mammalian nitro reductase system; however, NADPH is considerably more effective. The mammalian system is almost inactive in the presence of oxygen. Mammalian nitro reductase includes a flavoprotein component and is greatly stimulated by addition of excess flavin. A possible relationship between nitro reductase and the flavoprotein enzyme NADPH-cytochrome c reductase has been proposed by Kamm and Gillette (4), and a more recent study has suggested that 1 These investigations were carried out at the Western Fish Nutrition Laboratory, Cook, Washington. A portion of this work was presented at the annual meeting of the Federation of American Societies for Experimental Biology in Atlantic City, New Jersey, April, 1966 [Federation Proc. 26, 343 (1966)]. 2 Present address: Department of Agricultural Chemisty, Oregon State University, Corvallis, Oregon 97331.
cytochrome P-450 is a component of the mammalian nitro reductase system (5). Recently, Adamson and co-workers (6) detected azo and nitro reductase activity in the liver of two species of teleost fish. Hitchcock and Murphy (7), in addition, have recorded the reduction of nitro groups in several organic phosphorus insecticides by fish tissues. The present studies demonstrate the occurrence of an NADPH-dependent nitro reductase system in a number of fish species. The enzyme system is found mainly in liver tissue, where it is associated with the soluble fraction of the cell. The enzyme from fish liver has been characterized and found to be similar in many respects to mammalian nitro reductase, but it differs in its absence from the microsomal fraction. MATERIALS
AND
METHODS
Animals. Bluegill (Lepomis macrochirus), white crappie (Porno&s annularis), and yellow perch (Perca javescens) were obtained from a nearby lake on a hook and line. Carp (Cyrpinus carpio), Pacific lamprey (Lampetra tridentala), sockeye salmon (Oncorhynchus nerka), American shad (Alosa sapidissima), white sturgeon (Acipenser transmontanus), largescale sucker (Calostomus 582
REDUCTION
OF p-NITROBENZOIC
macrocheilus), and steelhead trout (Salmo gairdneri gairdneri) were trapped from the Columbia River at the Fish Passage Laboratory,8 U.S. Bureau of Commercial Fisheries, North Bonneville, Washington. Smallmouth bass (Micropterus dolomieui) , channel catfish (Zctalurus punctatus), flathead catfish (Pylodictis olivaris), and northern pike (Esoz Z&us) were obtained from the Miles City National Fish Hatchery, Miles City, Montana. Fingerling coho salmon (0. kisutch) and adult rainbow trout (S. gairdneri) were stock fish of the Western Fish Nutrition Laboratory, Cook, Washington. Fish were transported to the laboratory alive and maintained in large tanks until used experimentally. Freshly killed adult chinook salmon (0. tshawytscha) and coho salmon were collected immediately after spawning at the Spring Creek and Little White Salmon National Fish Hatcheries,’ Cook, Washington, and appropriate tissue samples were quickly transferred to the laboratory in cold isotonic KCl. Enzyme preparation. Fishes were exsanguinated, and livers and other tissue were removed immediately and homogenized in 2 volumes of cold 0.154 M KC16 with a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 10,OOOg for 20minutes, and the supernatant fractions, contain ing microsomes plus soluble fraction, were used for the majority of thestudies reported. Washedmicrosomes were prepared by centrifugation of the 10,OOOgsupernatantfractionat 105,OOOg for 60 minutes. After removal of the supernatant soluble fraction, the sedimented microsomes were suspended in fresh KCl, recentrifuged, and then resuspended in the original volume of 0.154 M KCl. Enzyme assays. Various fish enzyme preparations were incubated in duplicate with p-nitrobenzoic acid in a Dubnoff metabolic shaking incubator, usually for 2 hours at 25”. In most experiments the incubation mixture contained 1 ml of a tissue fraction derived from 0.34 gm fresh tissue, 0.5 Mmole NADP,” 1.25 rmoles MgCh, 10 rmoles glucose 6-phosphate, 1 Kornberg unit of glucose g-phosphate dehydrogenase (or 10 rmoles sodium isocitrate and 2 mg of isocitric dehydrogenase), 3 3 The cooperation of the late Mr. Joseph Cauley and his staff is gratefully acknowledged. 4 Arranged through the cooperation of Mr. Harlan Johnson. 5 Homogenization in potassium phosphate buffer, sucrose, or Tris-sucrose-KC1 gave preparations with somewhat lower nitro reductase activity. 6 Cofactors, enzymes, and additives to the incubation mixtures were products of the Sigma Chemical Co., St. Louis, Missouri.
ACID
BY FISHES
583
pmoles of p-nitrobenzoic acid, and 340 pmoles of potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Nitro reductase activity was determined at 540 rnp after trichloroacetic acid precipitation of protein, by diazotizing and coupling p-aminobenzoic acid to N-(l-naphthyl)ethylenediamine to yield a colored derivative as described by Fouts and Brodie (2). Appreciable quantities of p-aminobenzoic acid were also acetylated (2,s) by fish tissue preparations. The extent of conjugation was determined by heating aliquots of the trichloracetic acid supernatant in boiling water for 30 minutes (2, 8) and then diazotizing and coupling in the usual manner (2) to give the total (conjugated and free) p-aminobenzoic acid. Free paminobenzoic acid accounted for 30-35aj, of the total amine produced by liver 10,OOOgsupernatant and soluble fractions, and 45-50% by corresponding kidney fractions. There was no appreciable change in these ratios with pH, fish species, incubation temperature, cofactor and inhibitor concentration, or incubation time. The ratio of free to conjugated amine was relatively constant; consequently, the majority of incubation samples were only assayed for free p-aminobenzoic acid and these data are presented in this report. NADPH-cytochrome c reductase was assayed by following the changes in absorbancy at 550 rnr TABLE TISSUE
DISTRIBUTION
I
OF NITRO
ACTIVITY
REDUCTME
a
rmoles p-aminobenzoic acid fomedigm tissue/hour TiSSW
Liver Blood Muscle Anterior Posterior Heart Brain Spleen Testes
kidney kidney
Rainbow trout
Steelhead trout
Chinook salmon
0.230 0.002 Nil 0.023 0.120 Nil Nil 0.003 Nil
0.171
0.248
0.006 0.053
Nil
Nil 0.035 0.173 0.010 0.003 Nil
~1The incubation mixture contained 0.5 pmole NADP, 1.25 pmoles MgClz, 10 pmoles glucose g-phosphate, 1 Kornberg unit glucose 6-phosphate dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml 10,OOOg supernatant fraction from male fish (equivalent to 0.34 gm tissue), and 340 rmoles potassium phosphate buffer (pH 7.4), in a final volume of 5 ml. Incubations were carried out for 2 hours at 25” under nitrogen.
584
BUHLER TABLE
REQUIREMENTS ZOIC ACID
AND
II
FOR REDUCTION BY FISH LIVER
OF p-NITROBENPREPARATIONS~ ~moles -aminobenozic XL2 formed/gm liver/hour Rainbow Coho trout salmon
System
Complete Minus NADP Minus glucose 6-phosphate Minus Mgz+ Minus glucose B-phosphate dehydrogenase Plus air Minus all additives a Incubation conditions described in Table I.
0.168 0.032 0.102 0.164 0.158
0.191 0.07:)0.078 0.180 0.188
0.010
0.040 0.037
were the same as those
in a Beckman model DB spectrophotometer as described by Williams and Kamin (9). NADPHOxidase was determined spectrophotomet’rically at 340 rnp in the manner described by Gillette et al. (10). The glucose 6-phosphate dehydrogenase content of appropriately diluted fish liver preparations was measured by following the increase in absorbancy at 340 mp (11). Chemical assays. Lipid peroxidat,ion activit,y was measured by determining the amount of malonaldehyde formed with the thiobarbituric acid method of Ottolenghi (12). Lipid peroxide values have been expressed in terms of optical density at 530 rnp corrected for peroxide value due to the reagent blank.
RASMUSSON
activities were obtained on addition of NADP, glucose 6-phosphate, and a low concentration of MgClz (Table II). Enzyme activity was reduced appreciably by omission of NADP or glucose 6-phosphate and almost completely in an oxygen atmosphere. The rate of reduction of p-nitrobenzoic acid by trout supernatant fraction, 0.88 Imole of substrate reduced per gram liver per hour, was linear over the time interval examined (Fig. 1). Under aerobic conditions, however, t,he system rapidly lost activity. Some formation of p-aminobenzoic acid still occurred during the initial phases of the aerobic reaction, suggesting that the inhibition by oxygen was not an immediate process as one would expect if oxygen were acting as a competing electron acceptor, i.e., an NADPH-oxidase. Instead it appears that inhibition by oxygen occurred gradually, perhaps reflecting the slow oxidation of some sensitive component of the system. The effect of pH on nitro reductase activity is shown in Fig. 2. In phosphate buffer the optimum pH of the trout liver system was 7.4. The pH optima in pyrophosphate
RESULTS
Tissue distribution of enzyme activity. Incubation of p-nitrobenzoic acid with the 10,OOOgsupernatant7 fraction from various fish tissues showed that the liver and the posterior portion of the kidney were capable of reducing aromatic nitro groups (Table I). Anterior kidney and other fish tissues exhibited negligible activity. Previous studies in rabbits have shown that kidney tissue has one-half of the nitro reductase activity of liver (2). Requirements of the liver enzyme system. Requirements for the reduction of p-nitrobenzoic acid by fish liver supernatant fraction were similar to those reported for mammalian nitro reductase (2). Maximal 7 Homogenates pattern.
showed
essentially
the
same
0 0
1
2
3
4
Time IhoursI
FIG. 1. Reduction of p-nitrobenzoic acid by male rainbow trout liver supernatant fraction as a function of time. The incubation conditions were the same as those described in Table I and were carried out for the indicated time in air or nitrogen.
REIXJCTION
OF p-NITROBENZOIC
and Tris buffers were 8 and 7.6, respectively. Nitro reduct)ase activities in pyrophosphate or Tris buffers were about 70 and 30 % greater, respectively, than those observed with phosphate. .
ACID
,585
BY FISHES
Fish liver supernatant fraction which was heated for 2 minutes in boiling water and then cooled was completely inactivated. Frozen tissue samnles retained most of their activity, but subcellular preparations were partially inactivated by freezing or prolonged storage in the cold. The fish liver nitro reductase svstem was unusually sbable toward increases in temperature under the anerobic assay conditions (Fig. 3). Enzyme activity continued to increase with temnerature uruil 60” was reached. Above this temperature, the rate of reduction leveled off and then remained constant for incubation t,emperatures approaching 75”. Preincubation of the enzyme and cofactors at 25” in air in the absence of substrate, however, resulted in a substantial decrease in activity. When aerobic preincubation of fish liver enzyme was carried out for 1 hour at temneratures above 30”. enzyme activity was completely lost. There was no loss in nitro reductase activity if aerobic preincubation took place in 2 mM cysteine or in 10 rnM sodium pyrophosphate buffer, pH 7.4. Localization of enzyme activity. In contrast to the mammalian system (a), fish liver nitro reductase activity was not detected in microsomes but was confined to the 105,OOOg supernatant fraction (Table III). Significant microsomal activity could not be demonI
1
l,/
I
PH
FIG. 2. Relation of pH and nitro reductase activity with male rainbow trout supernatant preparation. Incubation conditions were the same as those described in Table I with 340 rrmoles of potassium phosphate, sodium pyrophosphate, or Tris buffer added as indicated.
2 c
5 .E 2
TABLE 0.300
INTRACELLULAR t
III
L~C.~LIZ.~TI~N
REDUCTMR
Intracellular fraction
-
pm&s p-aminobenzoicacid formed/gm liver/hour *T:t
01 0
I 20
I 40
I MI
I 80
Temperature hi
FIG. 3. Effect of incubation temperature on reduction of p-nitrobenzoic acid by male rainbow trout liver supernatant preparation. The incubation condit,ions were the same as those described in Table I except that incubations were carried out for 1 hour at the indicated temperatures.
Whole homogenate 10,000 gsupernatant fraction Microsomes Soluble fraction (103,OOOg supernatant fraction) Microsomes + trace solubleb
OF Nrrriro
ACTIVITY~
Coho salmon
Rainbow trout
0.444
0.166 0.159
0.190 0.186
0.021 0.326
0.004 0.161
0.008 0.145
0.057
0.032
0.024
y The incubation conditions were the same on those described in Table I. * Soluble fraction (0.1 ml) was added t.o 1 ml of the microsomal preparation.
586
BUHLER
AND
strated even with addition of a small quantity of the soluble fraction to the assay system. An appreciable sedimentation of nitro reductase activity was achieved by prolonged centrifugation of fish liver soluble fraction at 105,OOOgfor 8 hours (Table IV). Maximum activity was associated with a zone which also contained a flocculent reddish precipitate. An ultrafiltrate of fish liver soluble preparation was essentially devoid of nitro reductase activity (Table V), thus confirming the enzymic nature of the fish liver system. Enzyme activity was not lost by this treatment but instead remained concentrated in the residue from the ultrafiltration. Overnight dialysis of the soluble fraction against isotonic KC1 at 4” resulted in a significant loss of nitro reductase activity (Table V), while activity was retained in undialyzed material stored overnight under comparable conditions. Similar results were observed with the 10,OOOg supernatant fraction. The dialyzed enzyme could not be reactivated by addition of NADPH, flavin, or other cofactors. Addition of the ultraTABLE
IV
SEDIMENTATION OF FISH LIVER NITRO REDUCTASE ACTIVITY~ Fraction
Soluble fraction natant fraction) Upper aoneb Middle zoneb Bottom zoneb
(105,OOOg super-
rmoles p-aminobenozic acid formed/pm liver/hour 0.170 0.018 0.058 0.380
a The incubation conditions were the same as those described in Table I except that 1 ml of male coho salmon liver soluble fraction was employed. b The soluble (105,OOOg supernatant fraction) was centrifuged at 105,OOOg for 8 hours. The contents of the centrifuge tube were separated into three zones: upper (one-fourth of tube), containing a white flocculant suspension; middle (one-half of tube), clear yellowish zone; and bottom (one-fourth of tube), containing a reddish precipitate.
RASMUSSON TABLE V EFFECTS OF DIALYSIS AND ULTRAFILTRATION NITRO REDUCTASE ACTIVITJP Fraction
Soluble (105,OOOg supernatant)” Ultrafiltrate from solublec Residue from ultrafiltrationd Dialyzed soluble” Dialyzed soluble + ultrafiltrate from solublef Dialyzed soluble + cysteine (2 mM)
ON
fimoles of p-aminobenzoic acid formed’gm liver/hour 0.112 0.009 0.102 0.022 0.028 0.162
a The incubation conditions were the same as those described in Table I. This experiment used enzyme from male rainbow trout. b Stored overnight at 4”. c Prepared with a LKB Ultrafilter. d Reconstituted to the original volume with 0.154 M KCI. e Dialyzed overnight at 4” against 0.154 M KCl. ’ One ml of each preparation added to the incubation mixture.
filtrate from the soluble fraction also had little effect on the activity of the dialyzed preparation. Nitro reductase activity, however, could be restored to the original level or even stimulated by inclusion of cysteine in the incubation media or in the dialysis mixture. The reactivation by cysteine suggests that the loss of activity during the dialysis was probably an oxidative process. Dialysis apparently removed some low molecular weight components of the soluble fraction which normally protects the enzyme system against oxidative inactivation, and cysteine was able to substitute for these missing factors. Dialyzable factors in the soluble fraction have been reported to prevent the oxygen-induced respiratory decline of rat microsomes or mitochondria (13). Requirement for NADPH. NGtro reductase activity in the fish liver system was reduced significantly in the absence of added NADP or glucose 6-phosphate (Table II), indicating that the fish system required a reduced form of the coenzyme. The optimum concentration for added NADP was about 0.3 rmole (0.06 mM) (Fig. 4). Substitution of chemically reduced NADPH for the NADPHgenerating system gave a 30% stimulation
REDUCTION
OF p-NITROBENZOIC
in activity (Table VI), which is consistent with the requirement for reduced pyridine nucleotide. The isocitric dehydrogenase NADPH-generating system was somewhat more effective than the glucose g-phosphate dehydrogenase system, presumably because of the presence of glucose 6-phosphatase in the fish tissue preparations. Substitution of preformed NADH or a NADH-generating system was less effective than NADPH. Species diferences in enzyme activity. Table VII compares nitro reductase activity in liver preparations from various freshwater and anadromous fish species. No appreciable differences in enzyme activity as related to sex were noted in the limited number of samples tested; consequently the results were averaged without regard to the sex of the fish. Reduction of p-nitrobenzoic acid was greatest in preparations from the American shad and coarsescale sucker and least with the Pacific lamprey. Liver nitro reductase levels were comparable in juvenile and spawning adult silver salmon. The activities of liver preparations from most fish species were similar to that of the rat liver system assayed under the same conditions (Table VIII). Chan and co-workers (14) have reported a synergistic interaction of combined rat and trout liver preparations on cyclodiene
q,,
, 0
2 NADP Concentration
, 4
,
( 6
x 10m4M
FIG. 4. Effect of NADP concentration on the reduction of p-nitrobenzoic acid by male rainbow trout 10,OOOgsupernatant fraction. The incubation conditions were the same as those described in Table I except that the indicated amount of NADP was employed.
ACID
BY FISHES TABLE
REQUIREMENT
587 VI
FOR NADPHa
NADP (0.5 rmole), glucose B-phosphate (10 moles), and 1 unit glucose 6-phosphate dehydrogenase NADP (0.5 rmole), sodium isocitrate (10 pmoles), and 2 mg isocitric dehydrogenase NADPH (1 pmole) NADPH (3 pmoles) NADH (3 pmoles) NAD (0.5 pmole) and nn-malate (10 pmoles)
0.195
0.248
0.276 0.320 0.222 0.148
n The incubation mixture contained 1.25 pmoles MgC12, 3 pmoles p-nitrobenzoic acid, 1 ml male rainbow trout liver 10,OOOg supernatant fraction, and 340 pmoles potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Incubations were for 2 hours at 25” under nitrogen. b The mean of three such experiments are presented.
epoxidation. Incubation of fish and rat liver supernatant fractions together, however, gave a combined nitro reductase activity that was only a summation of the two individual activities (Table VIII). Total nitro reductase activity was more than doubled, however, when catfish and rainbow trout fractions were incubated together. Thus it appears that certain species of fish may exhibit a low nitro reductase activity because of a deficiency in some component of the enzyme system. This missing factor was apparently absent from rat liver preparations but could be supplied by the liver supernatant fraction from another fish species. The synergistic interaction of mixed fish preparations was largely eliminated when incubations were carried out in sodium pyrophosphate buffer (Table VIII), suggesting that the unknown fish factor and pyrophosphate have similar effects. The unknown component in fish preparations which stimulates the fish nitro reductase system may be related to the dialyzable protective agent in the soluble fraction previously described. Flavin requirements of the nitro reductase
585
BUHLER
AND
system. Mammalian liver nitro reductase has been shown to be a flavoenzyme by Fouts and Brodie (2), and Adamson and coworkers (6) have demonstrated the stimulatitin of shark liver nitro reductase by added flavin. The teleost liver nitro reductase system was also greatly stimulated by added flavin (Table IX). Flavin adenine dinucleotide (FAD), flavin mononucleotide TABLE RBDUCTION
VII
OF P-NITROBKNZOIC ACID VARIOUS FISH SPECIIB~
OF
pmoles of p-aminobenzoic acid formed.’ anl”yK
Fish speck&
Smallmouih bass (2) Bluegill (2) Carp (4) Channel catfish (4) Flathead catfish (2) White crappie (1) Pacific lamprey (8) Yellow perchc Northern pike (1) Chinook salmon (11) Coho salmon (14) Coho salmon fingerlings” Sockeye salmon (8) American shad (10) White sturgeon (8) Largesca!e sucker (7) Rainbow trout (20) SteeIliead trout (8)
IN LIVI~R
0.178 0.091 0.331 0.062 0.050
(2)
+ 0.006 f 0.009 f 0.053 f 0.009 f 0.017 0.072 0.019 f 0.003 0.072 f 0.019 0.143 0.253 f 0.037 0.177 f 0.043 0.228 f 0.018 0.312 f 0.092 0.485 f 0.098 0.082 f 0.027 0.559 & 0.073 0.198 f 0.039 0.183 f 0.044
a Metabolism expressed as mean pmoles of paminobenzoic acid formed per gram liver per hour f standard deviation), (each preparation was assayed in duplicate), averaged without regard to sex. The incubation mixtures contained 0.3 pmole NADP, 1.25 pmoles MgClz, 10 pmoles glucose g-phosphate, 1 Kornberg unit glucose 6phosphat’e dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml of liver 10,OOOgrupernatant fraction (equivalent to 0.34 gm liver), and 340 pmoles potassium phosphate buffer (pH 7.4) in a total volume of 5 ml. Incubations were for 2 hours at 25” under nitrogen; b Number of adult, animals per mean is ex-
pressed in parentheses. c Livers of several fish were pooled to obtain sufficient enzyme fo? assay. Mean of two such pools of adult yellow perch and fingerling silver salmon reported. Fingerling silver salmon averaged 20 gm per fish.
RASMUSSON TABLE NITRO
REDUCTUE SND RAT
VIII
ACTIVITY OF COMBINED ENZYME PREPARATIONS~
FISH
rmoles p-aminobenwic acid formed/gm liver/hour Fish enzyme
None Coho salmon 10,OOOgsupernat,ant fraction Rainbow trout 10,OOOg supernatant fraction Channel catfish 10,OOOg supernatant fraction Rainbow trout + Channel catfishsupernatant frac-1 tion
0.178
0.148” 0.285
0.160’
0.300”
0.030
0.3i4 0.128 0.585
a The incubation mixture contained 0.5 rmole NADP, 1.25 pmoles MgC12, 10 Fmoles sodium isocitrate, 2 mg isocitric dehydrogenase, 3 pmoles p-nitrobenzoic acid, 1 ml of fish liver supernatant fraction (equivalent to 0.34 gm liver), and 340 rmoles potassium phosphate buffer (pH 7.4) in a final volume of 5 ml. Incubations were carried out for 2 hours at 25’ under nitrogen. b Contained fish enzyme plus 1 ml of 0.154 M KC1 except in the mixed rainbow trout and channel catfish experiment. c Prepared from adult male Sprague-Dawley rats. One milliliter of supernatant preparation was added to the incubation mixture. d One milliliter of 0.05 M sodium pyrophosphate buffer (pH 7.4) was added to each incubation flask. e The average of two separate experiments.
(FMN), and riboflavin all enhanced activity, but FAD appeared to be most effective. Soluble and dialyzed soluble preparations also responded to flavins, albeit the magnitude of effect was considerably less. Soluble fraction was subjected to mild acid treatment to dissociate the flavin prosthetic group (15), and nitro reductase activity of the acid treated preparation was partially restored by added flavin. Acid treatment of the enzyme, however, caused an appreciable loss in activity. A similar susceptibility to denaturation upon mild acid treatment was reported for the azo reductance system from mammalian liver (16). In contrast, the activity of mammalain nits reductase was
REDUCTION TABLE EFFECT
OF p-NITROBENZOIC
IX
OF ADDED FLAVIN ON RAINBOW NITRO REDUCTASEO
TROUT
pm&s @minobenzoic acid formed/gm liver/hour Additive
None 0.01 mM FAD 0.1 mM FAD 1 mM FAD 0.01 mM FMN 0.1 mM FMN 1 mM FMN 0.01 mM Riboflavin 0.1 mM Riboflavin 0.7 mM Riboflavin
0.274 0.356 1.07 1.55 0.410 0.868 1.41
0.010
0.200
0.051
Nil
0.032 0.109
0.542 0.650
0.150
0.023 0.042
0.514 0.616
0.136
0.088
0.023 0.059
o.2601 I I I 0.405 1.32
(LThe incubation conditions were the same as those described in Table I. There was no detectable reduction of p-nitrobenzoic acid by an NADPH-generating system with added flavin in the absence of enzyme. b Soluble fraction was 105,OOOg supernatant fraction. c Dialyzed overnight at 4” against 0.154 M KCl. d Treated according to Zelitch and Ochoa (15).
ACID
Activation of fish nitro reductase by SH-type compounds is reminiscent of the bacterial nitro reductase system described by Saz et al. (17). The Escherichia coli required enzyme was NAD-dependent, cysteine or glutathione and Mn2+ for its activation, and was inhibited by low concentration of chlortetracycline. The fish and bacterial nitro reductase systems are not identical since the fish system was not inhibited significantly by chlortetracycline, nor was Mn2+ required for its activation. Moreover, EDTA stimulated the fish system but produced an inhibition of the bacterial enzyme, and the bacterial system retained appreciable activity under aerobic conditions while the fish enzyme did not,. Nicotinamide stimulated the mammalian system (a), but at a concentration of 20 rnn1 it caused a 40% inhibition of the fish liver nitro system (Table XI). reductase Nicotinamide also inhibited nitro reductase activity in washed fish liver microsomes and soluble and dialyzed soluble fractions. High concentrations of magnesium ions also produced a significant inhibition of the fish system in a manner analogous to that reported for mammalian enzyme (2). A slight stimulation in nitro reductase activity TABLE
not lost by such acid treatment, and added flavin completely reactivated the enzyme (2). Although washed fish liver microsomes produced only negligible reduction of p-nitrobenzoic acid, modest increases in activity were observed following incubation in the presence of high concentrations of flavin. E$ect of additives. In addition to flavin, a variety of other compounds stimulated the reduction of p-nitrobenzoic acid by fish liver preparations. The chelating agents EDTA, 8-hydroxyquinoline, and pyrophosphate increased nitro reductase activity, while chlortetracycline and cr,ar-dipyridyl were without effect (Table X). Nitro reductase activity was enhanced by addition of glutathione, 2-mercaptoethanol, or cysteine, the latter being most effective. Such sulfhydryl compounds were without affect on the mammalian enzyme (2).
589
BY FISHES
EFFECT
X
OF VARIOUS CHELATING AGENTS SULFHYDRYL COMPOUNDS ON VITRO REDUCTASE ACTIVITY~ Additive
Chlortetracycline . HCl LY,a-Dipyridyl Ethylenediaminetetraacetic acid (EDTA) 8-Hydroxyquinoline Sodium pyrophosphate, pH 7.4b Cysteine Glutathione Z-Mercaptoethanol
Concenn~~ion
AND
% S$n+3tion or mhlbltion
0.2 0.5 1
75 -9 +56
0.1 10
+114
2 2 2
+61 $29 $36
+21
a The incubation conditions were the same as those described in Table VIII. The assay mixture contained 1 ml of male rainbow trout liver 10,OOOg supernatant fraction (equivalent to 0.34 gm liver). b Potassium phosphate buffer was omitted from the incubation medium.
590
BUHLER TABLE
AND
XI
EFFECT OF VARIOUS INHIBITORS ON NITRO REDUCTASE ACTIVITY” Additive
M&L M8.L MgClz MgClz Nicotinamide Nicotinamide Nicotinamide Nicotinamide p-Chloromercuribenzoate p-Chloromercuribenzoate NaCN NaCN NaNB NaNs Carbon monoxide-nitrogenb Carbon monoxide (IOOY,) SKF-525A None, preincubated in air 15 min SKF-525A, preincubat,ed in air 15 min
Concentra-% ;;:i:tion (mx)
inhibition
0.25 1 5 15 1 5
+13 -7 -23 -34 -5
10
-25 -40 -22
20
0.1 1 0.1 1 0.1 1
1
-18
-80 -73 -91 -38 -36 -20 -40
-8 -55
1
-58
a The in:ubation conditions were the same as those described in Table VIII except that the indicated concentration of MgCl, was employed. The assay mixture contained 1 ml of male rainbow trout liver 10,OOOg supernatant fraction (equivalent, t,o 0.34 gm liver). b Carbon monoxide-nitrogen (1:4).
was observed at a magnesium concentration of 0.25 mM; therefore this level was selected for normal incubation conditions. The sulfhydryl binding agent p-chloromercuribenzoate (PCMB) almost completely inhibited the fish nitro reductase system (Table XI). Sodium cyanide and sodium azide also caused a significant inhibition of the system. Carbon monoxide reduced fish liver nitro reductase activity by 40%, suggesting the involvement of a carbon monoxide-sensitive hemoprotein component such as cytochrome P-450 (5). The microsomal oxidase inhibitor SKF-525A had little effect on nitro reductase activity. Although aerobic preincubation of the system with SKF-525A in the absence of substrate (18) did cause a sharp drop in activity, a similar loss occurred upon preincubation of the enzyme in the absence of
RASMUSSON
added SKF-525A. Decreased activity, therefore, must be ascribed to the oxygen-derived inactivation of the enzyme system as previously described. In addition, metal ions (Cuz+, Caz+, Mnz+, and Co2+) were without effect on nitro reductase activity at concentrations of 0.1 and 1 mM, while 0.1 mM Fe2+ caused a slight increase in activity. Lipid peroxidation. As measured by the thiobarbituric acid reaction, an appreciable peroxidation of lipids occurred in fish liver preparations (Table XII) in a manner similar to that previously reported for mammalian tissue (19, 20). Formation of lipid peroxide was greatest with washed microsomes and least in the soluble fraction.8 Addition of a trace of the soluble fraction to microsomes caused a significant reduction in the formation of thiobarbituric acid-reacting substances. A similar depression in microsomal peroxide formation by the soluble fraction has been reported for rat preparations (13). Lipid peroxide apparently accumulated during preparation of fish liver subcellular fractions since appreciable thiobarbituric acid-reacting material was detected at the start of the incubation period (Table XII). It is interesting to note that whereas this initial value increased with subsequent incubation in air, thiobarbituric acid color actually decreased following anerobic incubation. These results suggest that in the absence of oxygen some component of the incubation mixture was capable of reducing lipid peroxides or their decomposition products. Addition of pyrophosphate buffer to the incubation mixture caused a marked reduction in the thiobarbituric acid-reacting material derived from fish liver subcellular of fractions, and no further formation peroxides occurred when incubations were carried out in the presence of pyrophosphate or cysteine. Pyrophosphate has been previously found to inhibit lipid peroxidation in rat liver microsomes (21). Fish liver 10,OOOgsupernatant preparations contained five to six times as much glucose 6-phosphate dehydrogenase as did corresponding rat preparations. However, * The protein content of these fractions was not, determined, and lipid peroxidation might be more :omparable on a per milligram protein basis.
REDUCTION
OF p-NITROBENZOIC TABLE
INFLUENCE
OF VARIOUS AGENTS ON LIPID
ACID
XII
PEROXIDE FORMATION BY FISH LIVER
10,OOOg supernatant fraction 10,OOOg supernatant fraction 10,OOOg supernatant fraction 10,OGOg supernatant fraction Microsomes Soluble (105,OOOg supernatant fraction) Microsomes + trace solubleb
tion time (hours)
Air None
NaPPi
Cyst&e
NOW
0 0.5 1
0.122 0.174 0.167
0.069
0.112
0.062
0.122
0.122 0.115 0.101
2
0.176 0.550
1 1 1
PREPARATIONS?
ODrn
IDCUt%+
Fraction
591
BY FISHES
Nitrogen NaPPi
Cysteine
0.069
0.112
0.063
0.106
0.075 0.058
0.293
0.039 0.360
QThe incubation conditions were the same&sthose described in Table VIII. The reaction mixture contained 1 ml female rainbow trout liver preparation (equivalent to 0.34 gm liver). As indicated, 340 pmoles sodium pyrophosphate buffer (pH 7.4) (added in place of potassium phosphate buffer) or 10 pmoles cysteine were added to the incubation system. Incubations were carried out at 25” for the indicated time under either air or nitrogen. At the end of the incubation period, the reaction mixture was assayedfor lipid peroxide by the method of Ottolenghi (12). b Soluble fraction (0.1 ml) was added to 1 ml of the microsomal preparation. no significant correlation between fish liver glucose &phosphate dehydrogenase levels and nitro reductase activity was observed. Kamm and Gillette (4) have suggested that anerobic reduction of nitro compounds and aerobic oxidation of NADPH may be catalyzed by the same enzyme system (presumably NADPH-cytochrome c reductase), but no direct relationship between NADPH-oxidase and nitro reductase levels could be found in fish liver preparations. Fish liver microsomes contained little nitro reductase activity, although the NADPHcytochrome c reductase levels of microsomes was about eight times greater than that of the soluble fraction.9 A similar concentration of NADPH-cytochrome c reductase has been reported in liver microsomes from the rat (22) and rainbow trout (14). Overnight dialysis of the fish liver soluble fraction against 0.154 M KC1 resulted in a complete loss of endogenous cytochrome c reduction, but the level of NADPH-cytochrome c reductase remained unchanged. Dialysis of g The soluble fraction catalyzed an appreciable reduction of cytochrome c in the absence of added NADPH. In such cases, NADPH-cytochrome e reductase was determined by measuring the difference in the rates of reduction of cytochrome c in the presence and absence of added NADPH (22). The mean activity of rainbow trout liver microsomes was 7.2 units/gm liver, and that of soluble fraction was 0.9 unit/gm liver.
the soluble fraction had been found to produce an appreciable loss of nitro reductase activity (Table V). Effect of diet. No significant changes in liver 10,OOOg supernatant nitro reductase activity were found when groups of rainbow trout were starved for 1, 2, 4, and 8 weeks. These results are to be contrasted with those of Mato and Gillette (23), who observed significant increases in the nitro reductase activity of rat liver microsomes induced by starvation. Feeding rainbow trout diets high in protein or carbohydrate also failed to cause significant changes in liver nitro reductase levels. Oral administration or intraperitoneal injection of various inducing agents (phenobarbital, phenylbutazone, DDT, and chlordane) failed to produce a consistent increase in fish liver nitro reductase activity.lO lo Yearling rainbow trout were fed diets containing 10 or 25 mg technical or p,p’-DDT/lOO gm diet, and groups of fish were assayed at I, 2, and 4 weeks; were given daily intraperitoneal injections of corn oil containing 50 mg/kg technical DDT or chlordane for 4 days and assayed on the fifth day; or were given daily oral administration of 50 mg/ kg phenobarbital or phenylbutazone for 4 days and assayed.on.the fifth day. Fingerling coho salmon were given daily intraperitoneal injections of corn oil containing 50 mg/kg chlordane for 4 days and assayed on the fifth day.
BUHLER
AND
RASMUSSON
12
8 % x -I>
I/O -1
1 0
I
I
I
2
I
I
4
I 6
II rs1 x lo4 0
2.0
1.0
3.0
ml. Enzyme
FIG. 5. Effect of added enzyme on reduction of p-nitrobenzoic acid by male rainbow trout liver supernataht fraction. The incubation conditions were the same as those described in Table VIII with the addition of the indicated amount of male trout liver 10,OOOg supernatant fraction. Control experiment carried out in duplicate. Third experiment with 2 mM cysteine added to above incubation mixture.
Reaction kinetics. A plot of nitro reductase activity versus amount of rainbow trout liver supernatant enzymel’ was nonlinear at low enzyme concentrations (Fig. 5). Enzyme activity can be reduced in this characteristic manner at low enzyme levels if the enzyme preparation or incubation mixture contains a limited amount of a dissociable activator or coenzyme, or if an inhibitor of the system which could be gradually destroyed is present (24). The shape of the trout enzyme activity curve was not altered by omission of Mg2+ from t.he incubation medium. Substitution of NADPH for the NADPH-generating system or additions of lO- 4 M FNM failed to restore linearity, although the slope increased I1 Chinook and coho salmon 10,OOOgsupernatant preparations gave comparable results. Crude rainbow trout liver homogenates and soluble fraction behaved similarly.
FIG. 6. Double reciprocal plot of p-nitrobenzoic acid concentration against rate of reduction by male rainbow trout liver supernatant fraction. Velocities are given as moles/liter/hour, and substrate concentrations are in moles/liter. The incubation conditions were the same as those described in Table VII with the addition of the indicated amounts of p-nitrobenzoic acid to a total volume of 6 ml.
appreciably. Nitro reductase activity became directly proportional to enzyme concentration, however, when 2 lll~ cysteine was included in the incubation mixture (Fig. 5). As shown in Fig. 6, from the LineweaverBurk plot, the apparent K, value for the reduction of p-nitrobenzoic acid by rainbow trout supernatant fraction was about 1.03 X W4 M. Although the Michaelis constant for the mammalian nitro reductase system has not been reported, the Km for the trout liver system was about the same as those reported for the oxidation of various drugs by rat liver microsomes (25). Products of the reaction. An aliquot of the trichloroacetic acid filtrate from a typical incubation mixture was subjected to thinlayer chromatography on a silica gel GFl2 developed in butanol saturated with water. A single amine-containing compound with a mobility identical to that of authentic I2 Brinkman York.
Instrument
Co., Great Neck,
New
REDUCTION
OF p-NITROBENZOIC
p-aminobenzoic acid was detected after spraying the dried plate with diazotized sulfanilic acid. DISCUSSION
This investigation has shown that teleost fishes are capable of reducing p-nitrobenzoic acid by an NADPH-requiring enzyme system located primarily in the liver. Previous reports have indicated that the nitro reduct,ase system in mammalian liver is distributed between the microsomal and soluble portions of the cell (2). The fish enzyme, in contrast, was located predomiand nately in the soluble fraction, microsomes contained little activity. It is possible that the fish enzyme is actually localized in the endoplasmic reticulum in the intact cell but that it is more easily dissociated from the microsomes during isolation than is the mammalian system. The fish nitro reductase system, however, could be sedimented along with a very light particulate fraction when fish liver “soluble” enzyme was subjected to further centrifugation, suggesting that the fish nitro reductase system was associated with very light microsomes as previously described for the soluble mammalian enzyme (26). Mammalian azo reductase, which is similar in many respects to the nitro reductase system, is also found primarily in the soluble portion of the cell (16). The fish liver nitro reductase system was strongly inhibited by oxygen. This inhibition presumably reflected oxidation of some essential component of the system since liver preparations which were preincubated in air were no longer active. Some aerobic inactivation of the fish liver system apparent,ly occurred during preparation of the subcellular fract’ions and during the short period required for nitrogen to flush air completely from the incubator-shaker. Mammalian Intro reductase is also inhibited by oxygen (2), and recent studies have shown that preincubation of rat liver microsomes for 60 minutes under oxygen complet’ely destroyed nitro reductase activity while the system was completely stable under nitrogen (27). The oxygen derived inactivation of the fish nitro reduct,ase system may be related
ACID
BY FISHES
593
to the peroxidation of liver lipids. Mammalian liver preparations are known to undergo spontaneous lipid peroxidation (19, 20), and addition of hematin compounds (281, ascorbic acid (12), or heavy metals (12, 29) stimulates lipid peroxide formation by subcellular fractions. An NADPH-linked peroxidation of lipids in rat liver microsomes has also been reported (30, 31). Such lipid peroxides have a deleterious effect on many proteins and enzymes (19, 32, 33). Enzymes containing essential sulfhydryl groups appear to be especially susceptible to inactivation by lipid peroxides (33). Peroxidation of unsaturated fatty acids in the phospholipid containing microsomal membranes may be responsible for the inactivation of certain mammalian liver microsomal mixedfunction oxidases during aerobic incubation (34) * Antioxidants such as cysteine (33) and a-tocopherol (21, ZS), and chelating agents like EDTA and pyrophosphate (21)) prevent peroxidation of lipids in mammalian liver preparations. Cysteine also forestalled inactivation of the rat liver microsomal enzyme which hydroxylates napthalene (35). Other factors may be important, however, since ar-tocopherol failed to maintain the microsomal enzymes which metabolizes hexobarbital and codeine even though lipid peroxidation was abolished (34). Rainbow trout liver nitro reductase that had been inactivated by oxygen was restored to complete activity by addition of cysteine or sodium pyrophosphate. The trout enzyme was normally protected against oxygen inactivation by unknown dialyzable components of the soluble fraction (Table V). The relative instability of washed mammalian liver microsomal preparations is thought to be due t#o removal of such stabilizing factors (36). At low enzyme concentrations the fish liver nitro reductase system may be more susceptible to oxidative inact,ivation, as evidenced by the nonlinearity of the enzyme concentration-yield 1curve. Under these conditions limited amount,s of antioxidants in the soluble fraction apparently afford insufficient protection. Added cysteine, however, prevents oxygen damage and restores linearity to the enzyme concentration-yield curve (Fig. -5). The effectiveness
594
BUHLER
AND
of PCMB as an inhibitor of fish liver nitro reductase suggests that some component of the system contains essential sulfhydryl groups. One may speculate, therefore, that an initial peroxidation of lipids in fish liver preparations is followed by an inactivation of the nitro reductase system through oxidation of sensitive sulfhydryl groups (33). Cysteine and similar compounds probably serve as antioxidants and thus protect the system. Pyrophosphate may prevent inactivation of the nitro reductase system by chelating heavy metals which catalyze lipid peroxidation (12, 29), or it may stimulate the system by binding inhibiting magnesium ions (2) or maintaining NADPH levels through inhibition of endogenous pyrophosphatases (37). Nicotinamide is routinely added to the incubation mixture in most drug enzyme studies since it has been shown to stimulate reductase and mammalian liver nitro mixed-function oxidases (2, 38). Nicotinamide is thought to enhance drug metabolism in vitro by preventing the destruction of NADP by NADase (39). In fishes, however, nicotinamide has now been shown to be a good inhibitor of the liver nitro reductase system.13 In this connection, Schenkman et al. (40) have recently reported that nicotinamide does inhibit mammalian liver microsomal NADPH-dependent, mixedfunction oxidase, probably by interaction with cytochrome P-450. There is good evidence that the mammalian nitro reductase system contains cy$ochrome P-450 (5) and NADPH-cytochrome c reductase activity (4). The sensitivity of fish liver nitro reductase toward carbon monoxide (5) and nicotinamide (40) suggests that cytochrome P-450 may also be a constituent of the fish enzyme. In this connection, the cytochrome P-450 content of fish liver is known to be quite high (14, 41). Although NADPH-cytochrome c reductase may be involved in the mammalian nitro reductase system (4), fish liver micro13Nicotinamide also inhibits the liver microsomal mixed-function oxidase from fishes (D. R. Buhler and M. E. Rasmusson, Comp. Biochem. Physiol., in press).
RASMUSSON
somes which contain the majority of the NADPH-cytochrome c reductase are devoid of nitro reductase activity. In addition, the nitro reductase activity of the fish soluble fraction is greatly reduced by dialysis, whereas the NADPH-cytochrome c reductase activity remains unchanged. It seems apparent, therefore, that if NADPHcytoehrome G reductase is involved in the fish nitro reductase system, it is not the oxygen-sensitive or the rate-limiting component of the system. ACKNOWLEDGMENTS The authors acknowledge the diligent technical assistance of Miss Susan E. Anderson and Mrs. Donna R. Fink. We also thank Dr. Waldo S. Zaugg of the Western Fish Nutrition Laboratory, Cook, Washington, for his many helpful suggestions. REFERENCES 1. WESTFALL, B. B., J. Pharmacot. Exptl. Tbrup. 78, 386 (1943). 2. FOTJTS, J. R., AND BRODIE, B. B., J. Pharmacol. Exptl. Therap. 119,197 (1957). 3. MUELLER, W. A., 2. Physiol. Chem. 311, 155 (1958). 4. KAMM, J. J., AND GILLETTE, J. R., Life Sci., 2, 254 (1963). 5. GILLETTE, J. R., AND SASAME, H. A., Federation Proc. 24, 152 (1965). 6. ADAMSON, R. H., DIXON, R. L., FRANCIS, F. L., AND RALL, D. P., Proc. Natl. Acad. Sci. U.S. 64, 1386 (1965). 7. HITCHCOCK, M., AND MURPHY, S. D., Federation Proc. 26, 687 (1966). 8. HIETBRINK, B. E. AND DUBOIS, K. P., Radiation Res. 22, 598 (1964). 9. WILLIAMS, C. H., AND KAMIN, H., J. Biol. Chem. 237, 587 (1962). IO. GILLETTE, J. R., BRODIE, B. B., AND LADu, B. N., J. Pharmacol. Exptl. Therap. 119, 532 (1957). 11. KORNBERG, A., AND HORECKER, B. L., Methods Enzymol. 1, 323 (1957). 12. OTTOLENGHI, A., Arch. Biochem. Biophys. 79, 355 (1959). 13. GROVE, J. A., JOHNSON, R. M., AND CLINE, J. H., J. Biol. Chem. 241, 5564 (1966). 14. CHAN, T. M., GILLETT, J. W., AND TERRIERE, L. C., Comp. Biochem. Physiol. 20, 731 (1967). 15. ZELITCH, I., AND OCHOA, S., J. Biol. Chem. 201, 707 (1953). 16. FOUTS, J. R., KAMM, J. J., AND BRODIE, B. B., J. Pharmacol. Exptl. Therap. 120,291 (1957).
REDUCTION
OF p-NITROBENZOIC
17. SAZ, A. K., AND SLIE, R. B., Arch. Biochem. Biophys. 61,5 (1954). 18. GILLETTE, J. R., AND SESAME, H. A., Federation Proc. 23,537 (1964). 19. TAPPEL, A. L., AND ZALKIN, H., Arch. Biochem. Biophys. 80, 326 (1959). 20. Z~LKIN, H., AND TAPPEL, A. L., Arch. Biochem. Biophys. 88, 113 (1960). 21. CHATTERJEE, I. B., AND MCKEE, R. W., Arch. Biochem. Biophys. 110, 254 (1965). 22. PHILLIPS, A. H., AND LANGDON, R. G., J. Biol. Chem. 237, 2652 (1962). 23. KATO, R.,AND GILLETTE, J. R,. J. Pharmacol. Exptl. Therap. 160,279 (1965). 24. DIXON, M., AND WEBB, E. C. (eds.), “Enzymes,” Academic Press, New York (1964). 25. RUBIN, A., TEPHLY, T. R., AND MANNERING, G. J., Biochem. Pharmucol. 13, 1007 (1964). 26. FOUTS, J. R., Biochem. Biophys. Res. Commun. 6, 373 (1961). 27. GRAM, T. E., AND FOUTS, J. R., J. Pharmacol. Exptl. Therap. 162,363 (1966). 28. TAPPEL, A. L., AND ZALKIN, H., Nature, 186, 35 (1960). 29. MCKNIGHT, R. C., AND HUNTER, F. E., Biochim. Biophys. Acta 98, 643 (1965).
ACID
BY FISHES
595
30. HOCHSTEIN, P., AND ERNSTER, L., Biochem. Biophys. Res. Commun. 12, 388 (1963). 31. MAY, H. E., POYER, J. L., AND MCCAY, P. B., Biochem. Biophys. Res. Commun. 19, 166 (1965). 32. ROUBAL, W. T.,AND’TAPPEL, A.L., Arch. Biothem. Biophys. 113, 5 (1966). 33. WILLS, E. D., Biochem. Pharmacol. 7, 7 (1961). 34. GRAM, T. E., AND FOUTS, J. R., Arch. Biochem. Biophys. 114, 331 (1966). 35. BOOTH, J. AND BOYLAND, E., Biochem. J. 70, 681 (1958). 36. LEADBEATER, L., AND DAVIES, D. R., Biochem. PhurmacoZ. 13, 1607 (1964). 37. GILLETTE, J. R., GRIEB, W., AND SESAME, H., Federation Proc. 22, 366 (1963). 38. MITOMA, C., POSNER, H. S., REITZ, H. C., AND UDENFRIEND, S., Arch. Biochem. Biophys. 61, 431 (1956). 39. MANN, P. J. G., AND QUASTEL, J. H., Biochem. J. 36, 502 (1941). 40. SCHENKMAN, J. B., BALL, J. A. AND ESTABROOK, R. W., Biochem. Phurmacol. 16, 1071 (1967). 41. BRODIE, B. B., AND GILLETTE, J. R., American Chemical Society, Winter Meetings, 1966.