Interrelationships between ornithine,glutamate and GABA-III. an ornithine aminotransferase activity that is resistant to inactivation by 5-fluoromethylornithine

5-Fluoromethylornithine (5-FMOrn) is a specific inactivator of l-ornithine:2-oxoacid aminotransferase (OAT). However, a certain proportion of the OAT activity in mouse brain, liver and kidney is not inactivated by this compound. In the present work, the occurrence, distribution and subcellular localization of this 5-FMOrn-resistant OAT is reported. It was shown that the 5-FMOrn-resistant brain enzyme is kinetically different from the corresponding liver enzyme, and it also differs from the 5-FMOrn-sensitive OAT. The most conspicuous difference between the 5-FMOrn-resistant OAT of liver and brain is the sensitivity of the latter against excessive concentrations of its substrate 2-oxoglutarate.5-FMOrn and GABA are reversible inhibitors of the 5-FMOrn-resistant enzyme. Both compounds compete with Orn for the enzymes active site. A number of known inactivators of GABA-T which are at the same time inactivators of OAT, and canaline, a natural inhibitor of OAT, inactivate both the 5-FMOrn-sensitive and the 5-FMOrn-resistant enzyme. Gabaculine is the most potent inhibitor of the 5-FMOrn-resistant enzyme that is presently known. Our results are compatible with the suggestion that the 5-FMOrn-resistant OAT is an isoenzyme. From the fact that this form of OAT prevails in the brain, and its occurrence in the nerve ending fraction of brain homogenates supports the view that 5-FMOrn-resistant OAT may be involved in the intraneuronal generation of neurotransmitter glutamate and/or GABA from Orn as precursor. Further support in favour of this notion are previous findings which suggest feedback inhibition of OAT by GABA in GABAergic nerve endings.     

Interrelationships between ornithine,glutamate and GABA—I. Feed-back inhibition of ornithine aminotransferase by elevated brain GABA levels

In this work new methods for the determination of ornithine (Orn) and l-ornithine:2-oxoacid aminotransferase (OAT) activity are described. These methods were used to demonstrate linear interrelationships between brain GABA and Orn concentrations. Brain GABA levels were modulated by administration of vigabatrin (4-aminohex-5-enoic acid), a specific inactivator of GABA-T, which is not an inhibitor of OAT. The results suggest feed-back inhibition of OAT by GABA, a mechanism which is compatible with the assumption that Orn may serve in certain neurons as a precursor of glutamate and GABA.     

Ornithine aminotransferase activity, liver ornithine concentration and acute ammonia intoxication

5-Fluoromethylornithine (5FMOrn) is a specific inactivator of L-ornithine:2-oxoacid aminotransferase (OAT). Inactivation of OAT causes the enhancement of L-ornithine (Orn) concentrations in all tissues. Intraperitoneal or oral administration of 10-50 mg/kg of 5FMOrn per day to albino mice rendered partial protection against lethal intoxication with 26 mmol/kg of ammonium acetate. The protective effect was maximal around 16 h after 5FMOrn administration, at the time when endogenous Orn concentrations were maximal. At this time protection by 5FMOrn against acute ammonia intoxication was comparable to that observed 1 h after the intraperitoneal administration of 10 mmol/kg of L-arginine. Pretreatment with 5FMOrn prevented the enhancement of excessive urinary excretion of orotic acid by ammonia intoxicated mice, and it enhanced urea formation in the liver. These biochemical effects demonstrate that 5FMOrn shifts Orn into the urea cycle, Orn which normally would be transaminated. Since even long-term treatment of mice with 5FMOrn did not reveal toxic effects, this compound may be considered for the treatment of certain conditional deficiencies of Orn or arginine.     

Ornithine aminotransferase activity, tissue ornithine concentrations and polyamine metabolism

1. Inactivation of L-ornithine:2-oxoacid aminotransferase (OAT) by 5-fluoromethylornithine (5FMOrn), a specific inactivator of OAT, causes a great elevation of tissue ornithine (Orn) concentrations. 2. Inhibition of L-ornithine decarboxylase (ODC) by 2-difluoromethylornithine (DFMO) had no effect on Orn concentrations. 3. The combined administration of 5FMOrn and DFMO produced a 2- to 3-fold greater enhancement of tissue Orn concentrations than treatment with 5FMOrn alone. 4. The increase of tissue Orn concentrations had a long-lasting enhancing effect on polyamine metabolism. 5. In the brain this could be demonstrated by the elevation of putrescine and spermidine concentrations and the increase of spermidine turnover rate. 6. In visceral organs polyamine concentrations were not elevated because polyamines can be eliminated by transport. 7. In line with this notion is the fact that urinary polyamine excretion was increased for several days, even after a single dose of 5FMOrn. 8. Inhibitors of 4-aminobutyric acid:2-oxoglutarate aminotransferase which are also inactivators of OAT had the same effect on polyamine excretion as 5FMOrn.     

Effects of inhibition of ornithine aminotransferase on thioacetamide-induced hepatogenic encephalopathy

Repeated administration of thioacetamide (TAA) to CD1 mice produced hepatic failure and biochemical and behavioral effects characteristic of hepatogenic encephalopathy (HE). The symptoms in mice resembled those previously observed in rats after similar treatments. It is, howeve, obvious that both in rats and mice the severity of symptoms depends not only on dose and dosing schedule of TAA, but also on strain and body weight (age). Administration of 5-fluoromethylornithine (5FMOrn), a selective inactivator of ornithine aminotransferase (OAT), significantly reduced mortality, and it ameliorated most of the TAA-induced pathologic symptoms, such as hypothermia, decreased locomotor and exploratory behavior, pathologic liver function and amino acid patterns. The most prominent biochemical consequence of 5FMOrn administration is the elevation of ornithine concentrations in tissues, including the brain, and in body fluids. Elevated ornithine concentrations are, therefore, the most likely basis for the therapeutic effects of 5FMOrn. In agreement with this notion is the enhancement of citrulline and urea formation. These findings and the observation that administration of ornithine in combination with a branched-chain 2-oxoacid ameliorated the pathologic symptoms of portal-systemic encephalopathy suggest inhibition of OAT in the treatment of this disease. The liver protective effect of 5FMOrn is not yet understood; the enhancement of regenerative processes is a likely explanation.Abbreviations GABA 4-aminobutyrate - GABA-T 4-aminobutyrate aminotransferase - GOT plasma glutamate oxaloacetate transaminase - HE hepatogenic encephalopathy - LDH plasma lactate dehydrogenase - MAO monoamine oxidase - OAT ornithine aminotransferase - TAA thioacetamide - 5FMOrn 5-fluoromethylornithine Special issue dedicated to Dr. Claude Baxter.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

An invertase inactivator in maize endosperm and factors affecting inactivation

A protein present in the developing endosperm of maize (Zea mays L.) causes a loss of invertase activity under certain conditions of incubation. This protein, designated an inactivator, inactivates invertase I of maize even in the presence of other proteins. No inactivation of invertase II of maize or yeast invertase has been observed. The inactivator and invertase I are found only in the endosperm. The quantity of inactivator increases in the normal endosperm during development while invertase I activity decreases. However, the altered levels of invertase I activity in several endosperm mutant lines do not result from different quantities of inactivator. The inactivator can decrease invertase I activity during a preincubation period before addition of sucrose; inactivation is noncompetitive. Invertase I activity decreases curvilinearly with an increase in inactivator concentration. At high buffer concentrations or low inactivator concentrations in the reaction mixture, a latent period is observed when invertase I is not inactivated. Inactivation increases with an increase in temperature and a decrease in pH.     

DL-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase.

5-Fluoromethylornithine (5FMOrn) is an enzyme-activated irreversible inhibitor or ornithine aminotransferase (L-ornithine:2-oxo-acid 5-aminotransferase, OAT). For purified rat liver OAT, Ki(app.) was found to be 30 microM. and tau 1/2 = 4 min. Of the four stereomers of 5FMOrn only one reacts with OAT. The formation of a chromophore with an absorption maximum at 458 nm after inactivation of OAT by 5FMOrn suggests the formation of an enamine intermediate, which is slowly hydrolysed to release an unsaturated ketone. L-Canaline [(S)-2-amino-4-amino-oxybutyric acid] is a well-known irreversible inhibitor of OAT. Not only the natural L-enantiomer but also the D-enantiomer reacts by oxime formation with pyridoxal 5'-phosphate in the active site of the enzyme, although considerably more slowly. This demonstrates that the stereochemistry at C-2 of ornithine is not absolutely stringent. In vitro, canaline reacted faster than 5FMOrn with OAT. In vivo, however, only incomplete OAT inhibition was observed with canaline. Whereas intraperitoneal administration of 10 mg of 5FMOrn/kg body wt. to mice was sufficient to inactivate OAT in brain and liver by 90% for 24 h, 500 mg of DL-canaline/kg body wt. only produced a transient inhibition of 65-70%. The accumulation of ornithine in these tissues was considerably slower and the maximum concentrations lower than were achieved with 5FMOrn. It appears that DL-canaline, in contrast with 5FMOrn, is not useful as a tool in studies of biological consequences of OAT inhibition.     

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

The ornithine aminotransferase gene in gyrate,atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts

Summary Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2, cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from, a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern, of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i. e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene. This project was initiated in the laboratory of H. C. P. and was supported by grants CA07175, CA22484, and 5 T32 CA09020 from the National Cancer Institute and Postdoctoral Fellowship PF-2414 from the American Cancer Society. The continuing work in the laboratory of J. D. S. was supported by grants CA36727 and HD24189 from the National, Institutes of Health, grants SIG-16, ACS-IN165A, and a Junior Faculty Research Award (JFRA-227) from the American Cancer Society, and by University of Nebraska Medical Center Seed Research Grant 88-10.     

A New Synaptosomal Biosynthetic Pathway of Glutamate and GABA from Ornithine and Its Negative Feedback Inhibition by GABA

In sonicates of mouse brain synaptosomes, we demonstrated that gamma-aminobutyric acid (GABA) can be formed when L-ornithine (Orn) through L-glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]L-proline (Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1-5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine delta-aminotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30-fold-purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, L-glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or beta-alanine. The inhibition of GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Orn is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of L-glutamic-gamma-semialdehyde in equilibrium with L-delta 1-pyrroline-5-carboxylic acid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It is confirmed that L-canaline (delta-aminooxy-L-alpha-aminobutyric acid) is a potent and specific inhibitor of brain OAT whereas much weaker inhibition was observed with two other carbonyl-trapping agents, aminooxyacetic acid and hydrazine.     

Ornithine delta-aminotransferase activity in retina and other tissues

Ornithine -aminotransferase (OAT) activity was determined in liver, kidney, brain, retina and ciliary body-iris of rat, rabbit, calf and human. OAT activities (nanomoles ¹-pyrroline-5-carboxylate/mg protein/hr) in retina were (mean±SE) 324±43, 240±24, 234±26 and 218±22 respectively in rat, rabbit, calf and human. The OAT activities in retina were three times higher than in brain and 80% of that of liver. 2-oxoglutarate was the preferred amino acceptor substrate for OAT activity. In rat retina the activities of OAT with glyoxalate, -hydroxypyruvate, pyruvate, and oxaloacetate were 51, 44, 30, and 30% of that of 2-oxoglutarate respectively. A lack of substrate OAT specificity indicates OAT deficiency such as occur in gyrate atrophy of the choroid and retina could impair metabolism of ketoacids. A candidate for possible toxicity to the retina in OAT deficiency is glyoxalate. Arginine glycine transamidinase activity was not detectable in human retina, thus a previously postulated creatine phosphate deprivation in OAT deficiency may not be applicable to the pathogenesis of the disease.     

Interrelationships between ornithine,glutamate, and GABA. II. Consequences of inhibition of GABA-T and ornithine aminotransferase in brain

The objective of the present study was to compare the effects of elevation of GABA concentration and those of inactivation ofl-ornithine: 2-oxoacid aminotransferase (OAT) on the in vivo metabolism ofl-ornithine (Orn) in brain. Vigabatrin (4-aminohex-5-enoic acid) and gabaculine (5-amino-1,3-cyclohexadienyl carboxylic acid), two well known inactivators of GABA-T, were used to elevate brain GABA concentrations. The latter inactivates OAT also. Transamination of Orn is, from a quantitative point of view, a significant reaction in mouse brain. GABA is a feed-back regulator of OAT. Within GABAergic neurons Orn concentration may be regulated by endogenous GABA. Extensive inactivation of OAT causes a considerable increase of Orn concentration, both in synaptosomes and in non-synaptosomal compartments. The results are compatible with a role of Orn as precursor of glutamate and/or GABA in certain neurons.     

Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵¹-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.     

Influence of mevalonate kinase on studies of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The nature of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase has been studied. Several observations suggest that reductase inactivator preparations from both microsomes and cytosol possess mevalonate kinase activity. (1) Reductase inactivator (reductase kinase) activity copurified with mevalonate kinase activity. (2) Inactivator activity was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, known to be potent inhibitors of mevalonate kinase. (3) Addition of an excess of mevalonate completely prevented inhibition of reductase activity. (4) Formation of phosphomevalonate fully accounted for the decreased amount of mevalonate formed in the presence of inactivator and MgATP. (5) When reductase activity was measured by NADPH oxidation, no inhibition was observed. Clearly, the presence of mevalonate kinase in reductase inactivator preparations can lead to misinterpretations concerning whether reductase activity is regulated by phosphorylation-dephosphorylation. In this paper, we present several methods and approaches which can be used to critically evaluate this possibility.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.