The oxidation of indoe-3-acetic acid by anionic tomato peroxidase was found to be neglUige unless reaction mixtures were supplemented with H202. The addition of H202 to reaction mixtures initiated a period of rapid indole-3-acetic acid oxidation and 02 uptake; this phase ended and 02 uptake fell to a low level when the H202 was exhausted. The stoichiometry of the reaction, which is highly dependent on enzyme concentration and pH, suggests that H202 initiates a sequence of reactions in which indole-3-acetic acid is oxidized.Many plant peroxidases catalyze the oxidation of IAA by molecular 02 in the presence of a phenol and manganous ion (2,6,10,14,16 MATERIALS AND METHODSEnzyme Source and Estimation. Tomato anionic peroxidase was extracted from tomato (Lycopersicon esculentum var. tropic) pericarp tissue and purified to about 85% homogeneity as previously described (11). Enzyme concentrations were estimated from the 403 nm A of solutions, using a millimolar extinction coefficient of 107 for the enzyme (11). A spectra from 400 nm to 600 nm wavelength were run on a Perkin-Elmer 124 spectrophotometer and associated chart recorder and used to determine if the enzyme was present as ferric enzyme, ferrous enzyme, compound I, compound II, or compound III (11

A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.A single peroxidase which has been shown to exist in tomato fruit extracts and to exhibit some IAA oxidase activity (5, 8) has been implicated both in the production of ethylene (12,17,18) and in the destruction of the plant growth hormone IAA (8). Although it is not unusual to relate the action of the enzyme peroxidase with the control of the above hormones, in the case of tomato fruit it is premature to assume such a relationship because of insufficient information about the physical and catalytic properties of this peroxidase and the lack of a purification method which would allow a quantitative estimation and a complete isozyme composition of the noncovalently bound peroxidases of the fruit (22, 23).Spectral properties and pH optima of the tomato fruit peroxidase in catalyzing the oxidation of redogenic substrates in the presence of H202 have been previously reported by Evans (6). The present report deals with additional properties of the purified enzyme including its capability to be converted to complexes of higher oxidation states (compounds I, II, III) which are thought to be important for the IAA oxidase activity of peroxidases (13,26). The enzyme HRP,3 a well studied peroxidase, was employed in order to compare some of its properties with similar properties of the tomato enzyme. ' cheesecloth, and the residue was washed twice with 100 ml of 0.1 M phosphate buffer (pH 6.5). The filtrates were combined and centrifuged at 2,000 g for 10 min. The supernatant, to be referred to as crude soluble peroxidase fraction, was stored at 0 C for further use. The pellet and the solid material on the cheesecloth were suspended in 200 ml of 0.2 M sodium maleate-0.2 M calcium chloride adjusted to pH 6.5. The suspension was then sonicated for 5 min and centrifuged at 2,000g for 10 min. Under this treatment peroxidases that were ionically bound to the tomato pulp were solubilized (17). These per...

Hamsters and rats metabolize [1-14C]N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitrosobis(2-oxopropyl)amine (BOP) to yield N-nitrosobis(2-hydroxypropyl)-amine (BHP), glucuronic acid conjugates of HPOP and BHP, the sulfate ester of HPOP and 14C-labeled urea, all of which are excreted, and 14CO2 which is both incorporated in the urea cycle, and exhaled. The extent of metabolism and the ratios of these metabolites does not vary significantly with age or sex of the animal, however, marked species differences are evident in the metabolite composition of urine 6 h following administration of HPOP. Hamsters sulfate HPOP several times more rapidly, and reduce it to BHP more efficiently than rats. In contrast, the rat excretes more unchanged HPOP and its glucuronic acid conjugate than the hamster. Since sulfation and glucuronidation of HPOP may be involved in its activation and detoxication, these reactions were examined in detail in order to elucidate the reason(s) for their distinctively different contributions to its metabolism in rats and hamsters. Conjugation of HPOP with glucuronic acid and sulfate occurs in the livers of both rats and hamsters and is catalyzed by microsomal glucuronyl transferases and cytosolic sulfotransferases, respectively. The levels of glucuronyl transferase activity for conjugating phenolic compounds are comparable in the livers of two species; however, glucuronidation of HPOP is catalyzed by an isozyme the activity of which is three times greater in rat than in the hamster. In contrast to glucuronidation, sulfation of HPOP is catalyzed approximately 10 times faster by hamster than rat liver cytosol. Although rat liver can catalyze sulfation of phenolic compounds very effectively, it has low activity in sulfating aliphatic alcohols and beta-hydroxynitrosamines. Since both aliphatic alcohols and HPOP are sulfated by hamster liver cytosolic preparations and since these reactions are not significantly affected by the classic phenol sulfotransferase inhibitors, it appears that beta-hydroxynitrosamines may be sulfated by the aliphatic (hydroxysteroid) sulfotransferase isozymes. The failure of the rat to extensively sulfate HPOP in vivo may be attributed to the high Km of rat hydroxysteroid sulfotransferases for this compound. Of the four isomers of HPOP, only isomer A, in which the nitroso group is syn to the free keto group, is sulfated in vitro to an appreciable extent. The other three isomers either are not sulfated, or become unstable and decompose when they undergo such a reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

It is generally held that altered areas, neoplastic nodules and hepatocellular carcinomas (HCC) induced by mutagenic chemical carcinogens are resistant to the effects of hepatotoxins. This characteristic is attributed to the marked decrease in activating (phase I) enzymes and a several-fold increase in detoxifying (phase II) enzymes. In previous studies, we have shown that hepatic neoplastic lesions induced by non-mutagenic peroxisome proliferators differed from mutagenic carcinogen-induced lesions by lacking gamma-glutamyl transpeptidase and the placental form of glutathione S-transferase. In this study we have examined ciprofibrate-induced HCC for phase I and phase II enzymes. These tumors showed a marked decrease in cytochrome P-450 (53%), cytochrome b5 (79%) and aryl hydrocarbon hydroxylase (55%) activities compared to normal livers. Interestingly, activities of phase II enzymes in these tumors, such as UDP-glucuronyltransferases and sulfotransferases were decreased or remained the same as in the normal livers. In addition, the activity of epoxide hydrolase was also decreased markedly in all peroxisome proliferator-induced HCC. The decrease in the activity of various enzymes appears not to be due to the direct effect of ciprofibrate, since no inhibitory effect was observed after adding this compound in vitro. These findings further amplify the differences between the hepatic lesions induced by mutagenic hepatocarcinogens and non-mutagenic peroxisome proliferators suggesting a divergence in the mechanism by which peroxisome proliferators induce liver tumors.

Liver microsomes from male Syrian golden hamsters and Sprague Dawley rats metabolize the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine (NNDM) to N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product detectable by h.p.l.c. The rates of total metabolism are similar for both the cis and trans isomers; but the cis isomer of NNDM yields greater than 70% of the total product as HPOP while the trans isomer yields HPOP only as a minor product (20-30%) in both hamster and rat. The inability to identify other products could be attributed to alpha-hydroxylation which leads to fragmentation of NNDM and loss of tritium label to water. In order to investigate the possibility of the participation of an alpha-hydroxylation reaction, the metabolism of NNDM fully deuterated at either the 3 and 5 (alpha-d4) or the 2 and 6 (beta-d2) positions was examined and compared to the metabolism of the undeuterated compound (d0). Although the rates of metabolism of all the cis and trans derivatives of NNDM were similar (VMax = 2.13 nmol/min/mg hamster microsomal protein) as determined from measurements of substrate disappearance, the yields of HPOP were different. Maximum HPOP yields were observed with cis alpha-(d4) NNDM (93.9% of the total), followed by cis d0 NNDM (72.3%), trans alpha-(d4) NNDM (60.1%), trans d0 NNDM (30.2%), cis beta-(d2) NNDM (19.5%) and trans beta-(d2) NNDM (8.5%). These results suggest that alpha-hydroxylation is an alternative to beta-hydroxylation. Since the carcinogenic potency of the various deuterium derivatives of NNDM for the Syrian golden hamster parallels their ability to yield HPOP, beta-hydroxylation is closely related to pancreatic carcinogenesis in the hamster. Rat liver microsomal fractions showed the same patterns of HPOP formation to total metabolite yields as hamster liver microsomes with both the cis and trans isomers. However, rates of NNDM metabolism and HPOP formation were 7 times faster with hamster than with rat liver microsomes. Such a difference may be related to the failure of the cis isomer to induce pancreatic cancer in rats.