Hydrogen sulfide attenuates intracellular oxidative stress via repressing glycolate oxidase activities in Arabidopsis thaliana.

BACKGROUND: Hydrogen sulfide (H2S) has been proposed to exert anti-oxidative effect under many environmental stresses; however, how it influences oxidative stress remains largely unclear. RESULTS: Here, we assessed the effects of H2S on oxidative stress responses such as salicylic acid (SA)-dependent cell death, which triggered by increased H2O2 availability in Arabidopsis thaliana catalase-deficient mutants cat2 displaying around 20% wild-type catalase activity. H2S generation and its producing enzyme L-cysteine desulfhydrase (LCD/DES) were found to transient increase in response to intracellular oxidative stress. Although introducing the mutation of des1, an important LCD, into the cat2 background produced little effect, H2S fumigation not only rescued the cell death phenotype of cat2 plant, but also attenuated SA accumulation and oxidation of the glutathione pool. Unexpectedly, the activities of major components of ascorbate-glutathione pathway were less affected by the presence of H2S treatment, but decreased glycolate oxidase (GOX) in combination with accumulation of glycolate implied H2S treatment impacts the cellular redox homeostasis by repressing the GOX-catalyzed reaction likely via altering the major GOX transcript levels. CONCLUSIONS: Our findings reveal a link between H2S and peroxisomal H2O2 production that has implications for the understanding of the multifaceted roles of H2S in the regulation of oxidative stress responses.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Aldo-keto reductase-7A protects liver cells and tissues from acetaminophen-induced oxidative stress and hepatotoxicity.

UNLABELLED: Aldo-keto reductase-7A (AKR7A) is an enzyme important for bioactivation and biodetoxification. Previous studies suggested that Akr7a might be transcriptionally regulated by oxidative stress-responsive transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a protein highly responsive to acetaminophen (APAP) or its intermediate metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). This study was, therefore, carried out to investigate whether Akr7a is involved in the protection against APAP-induced oxidative stress and hepatotoxicity. We found that in response to APAP or NAPQI exposure, Akr7a3 mRNA and protein were significantly up-regulated in vitro in human HepG2 and LO2 cells. Similarly, strong induction was observed for Akr7a5 in mouse AML12 hepatocytes exposed to APAP. In vivo in wild-type rats, significant up-regulation of hepatic AKR7A1 protein was observed after administration of APAP. On the other hand, depletion of Nrf2 reduced the expression of Akr7a3, suggesting that Nrf2, indeed, contributes significantly to the induction of Akr7a. Moreover, loss of cell viability in Nrf2-depleted cells was significantly rescued by coexpression of AKR7A3. Furthermore, increased AKR7A3 in HepG2 cells was associated with the up-regulation of oxidative stress-related enzymes to enhance cellular antioxidant defense, which appeared to contribute significantly to protection against APAP-induced toxicity. In a line of transgenic rats overexpressing AKR7A1, increased AKR7A1 stimulated the expression of Nrf2 and other Nrf2-regulated genes, but did not better protect rats from APAP insults. In contrast, depletion of Akr7a5 in vitro in cultured AML12 cells or depletion of Akr7a1 in vivo in rat liver greatly increased APAP-induced hepatotoxicity. CONCLUSION: AKR7A proteins are significantly up-regulated in response to APAP/NAPQI exposure to contribute significantly to protection against APAP-induced hepatotoxicity. AKR7A mediates this protection, in part, through enhancing hepatocellular antioxidant defense.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFDelta1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFDelta1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFDelta1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFDelta1-480. Therefore, AIFDelta1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFDelta1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFDelta1-480. Human Jurkat cells transfected with the immuno-AIFDeltal-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFDeltal-480 gene as a novel approach to treating HER2-overexpressing cancers.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

Competing roles of cytochrome P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (+/-)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts.

(+/-)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol), a proximate carcinogen derived from benzo[a]pyrene (BP) requires further metabolic activation to exert its carcinogenic effects. Two principal pathways have been implicated, and these involve either the formation of (+/-)-trans-7,8-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) catalyzed by P450 1A1/P450 1B1 (NADPH-dependent monoxygenases) or the formation of benzo[a]pyrene-7,8-dione (BP-7,8-dione) catalyzed by human aldo-keto reductases AKR1A1 and AKR1C1-AKR1C4 [NAD(P)(H)-dependent oxidoreductases]. The relative contributions of the two pathways to PAH activation are unknown. In this study, BP-7,8-diol metabolism was studied in human bronchoalveolar H358 cell extracts. Parental H358 cells do not constitutively express P450 1A1/P450 1B1 or AKRs but were manipulated by induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to express P450 1A1/P450 1B1 or by stable transfection to express AKR1A1 (aldehyde reductase). TCDD induction of AKR1A1 transfectants provided a cell line that expressed both pathways. Extracts derived from parental H358 cells plus TCDD (P450 induction) produced electrophilic anti-BPDE, which hydrolyzed to benzo[a]pyrene tetrahydrotetrols (BP-tetrols), extracts derived from AKR1A1-transfected cells (AKR1A1 expression) produced reactive and redox-active BP-7,8-dione, which was trapped in situ as its mono(thioether) conjugate, and extracts derived from AKR1A1 transfectants plus TCDD (coexpression of P450 1A1/P450 1B1 and AKR1A1) produced both anti-BPDE and BP-7,8-dione. The competing activation of BP-7,8-diol by P450 1A1/P450 1B1 and AKR1A1 was studied with varied NADPH:NAD+ ratios. The system with a relatively higher concentration of NADPH favored formation of anti-BPDE via P450 1A1/P450 1B1, while the system with the higher concentration of NAD+ favored formation of BP-7,8-dione via AKR1A1. Under conditions that mimic the cellular redox state, 10 microM NADPH and 1 mM NAD+, equal amounts of BP-tetrols and BP-7,8-dione were formed. This suggests that P450 1A1/P450 1B1 and AKR1A1 play competing roles in the metabolic activation of BP-7,8-diol and that the dominant pathway of BP-7,8-diol activation depends on the redox state of the cells. These model systems provide a cellular context in which the dominant DNA adducts/lesions formed by either pathway may be compared.

Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo-keto reductase isoforms: potential antineoplastic agents that work independently of cyclooxygenase isozymes.

Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPARgamma). In human myeloid leukemia (HL-60) cells, ligand access to PPARgamma is regulated by AKR1C3, which diverts PGD(2) metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPARgamma-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC(50), K(I) values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC(50) values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH(2)(+) and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

The roles of aldo-keto reductases in steroid hormone action.

The human aldo-keto reductase 1C (AKR1C) isozymes are implicated in the pre-receptor regulation of steroid receptors, nuclear orphan receptors and membrane-bound ligand-gated ion channels. Human AKR members that may regulate the local concentration of steroid hormones include: AKR1C1, AKR1C2, AKR1C3, AKR1C4 and AKR1D1. Since, these enzymes are pluripotent, the physiological role for the human AKR1C isozymes is determined by their tissue-specific expression patterns and their substrate availability in target tissues. AKRs work in concert with short-chain dehydrogenases/reductases as switches to control ligand access to nuclear receptors. Consequently, they are potential targets in treating prostate cancer, breast cancer, endometriosis and endometrial cancer.

Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines.

We previously investigated the correlations between the expression of 9216 genes and various chemosensitivities in a panel of 39 human cancer cell lines(1)) and found that the expression levels of AKR1B1 and CTSH were correlated with sensitivity and resistance to multiple drugs, respectively. To validate these correlations, we investigated the expression of these two genes and the chemosensitivities in 12 additional gastric cancer cell lines. The expression of AKR1B1 in the additional cell lines exhibited significant correlations with sensitivities to 8 of the 23 drugs examined, while that of CTSH displayed a significant negative correlation with only one (MS-247) of the 27 drugs examined. Their expressions were weakly correlated with sensitivity and resistance, respectively, to the remainder of the drugs. Moreover, when the 12 cell lines were divided into high-expressing and low-expressing groups, a comparison of these groups using Mann-Whitney's U test revealed that high expression levels of AKR1B1 and CTSH were related to sensitivity to 21 of the drugs and resistance to 8 of the drugs, respectively. The present results suggest that AKR1B1 and CTSH may be good markers for prediction of sensitivity to certain drugs and that our panel of 39 cell lines has the potential to identify candidate predictive marker genes.

Cadmium exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity.

Administration of cadmium (Cd) at a dose of 1.23 mg/kg (2.0 mg/kg as CdCl(2)) markedly decreased the activity of an enzyme (acetohexamide reductase) catalysing the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats. However, the decreased enzyme activity was increased by repeated treatment with testosterone propionate (TP). When male rats were castrated and TP was given to the castrated ones, a similar decrease and increase, as described above, were observed in the microsomal enzyme activity. Cd exposure to male rats induced haemorrhage and atrophy of the testes and significantly diminished serum testosterone levels. There was no possibility that Cd accumulated in liver microsomes of male rats causing direct inhibition of the microsomal enzyme activity. We conclude that Cd exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity. Cd exposure had no effect on acetohexamide reductase activity in liver cytosol of male rats.

Alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium-treated rats.

We examined the alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium (Cd)-treated rats. Acetohexamide reductase activity in kidney microsomes of male rats was markedly decreased by treatment with Cd at a dose of 1.23 mg/kg body weight. However, the decreased enzyme activity was completely restored by repeated treatment with testosterone propionate. Therefore, it is reasonable to assume that the treatment with Cd indirectly affect the androgen-dependent acetohexamide reductase activity in kidney microsomes of male rats, possibly by depressing androgen production. In the case of female rats, unlike male rats, the microsomal enzyme activity was little detectable, and was unaffected by the treatment with Cd. Furthermore, Cd treatment had no significant effect on acetohexamide reductase activity in kidney cytosol of male or female rats.

Regulation of poly(beta-hydroxybutyrate) synthesis in Methylobacterium rhodesianum MB 126 growing on methanol or fructose.

The intracellular concentration of CoA metabolites and nucleotides was determined in batch cultures of Methylobacterium rhodesianum grown on methanol and shifted to growth on fructose. The intracellular concentration of CoA decreased from a high value of 0.6 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass during growth on methanol to a low value of 0.03 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass after a shift to fructose as a carbon source. The levels of NADH, NADPH, and acetyl-CoA were also lower. Under these conditions, acetyl-CoA was metabolized by both citrate synthase and beta-ketothiolase, and poly(beta-hydroxybutyrate) synthesis and growth occurred simultaneously during growth on fructose. Moreover, the level of ATP was approximately 50% lower during growth on fructose, supporting the hypothesis of a bottleneck in the energy supply during the growth of M. rhodesianum with fructose.

Protein stabilisation using additives based on multiple electrostatic interactions.

A method of elevating the storage lifetime of purified proteins has been discovered which appears to confer stability to all proteins investigated and may therefore be classed as generic in action. The basic methodology involves the formation of multiple electrostatic complexes between the protein and selected soluble polyelectrolytes to give protein-polyelectrolyte (PP) complexes and then to add solutions of polyalcohols or other compounds containing multiple hydroxyl groups. Dehydration of the resulting solution by vacuum evaporation, freeze drying or forced air convection produces a dry film or powder of stabilised protein. The method has been used mainly in the preparation of active enzymes for analytical tests. It has also been found that the formation of PP complexes also enhances the stability of enzymes in solution and the technique may be applicable to the stabilisation of virus suspensions by polycations. Examples of stabilised enzymes prepared by these methods are given and the proposed mechanism of stabilisation and applicability of the method to shelf-stable vaccine products are discussed.

Sublethal effects of inorganic mercury on the body growth rate and liver function enzymes of phenobarbitone-pretreated and promethazine-pretreated rabbits.

Hepatotoxic effects of inorganic mercury with and without pretreatment of phenobarbitone and promethazine have been described in experiments on domesticated rabbits. The total body weight and the relative liver weight decreased after mercury treatment under all experimental conditions. After phenobarbitone (PB) treatment, the serum glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), isocitrate dehydrogenase (ICDH), and lactate dehydrogenase (LDH) activities decreased to 31%, 77%, 20%, and 27%, respectively, whereas the serum alkaline phosphatase (AP) activity increased 54%. After promethazine (PM) treatment, however, the serum GPT activity was inhibited 73%, whereas the serum LDH activity increased 53%. Both hepatic GPT and AP activities decreased after PB (41% and 46%, respectively) and after PM (50% and 52%, respectively) treatments, while the activities of LDH and ICDH increased (after PB: 924% and 108%, respectively; after PM: 147% and 40%, respectively). After mercuric chloride (HgCl2) treatment, the serum GOT, GPT, LDH, and ICDH activities decreased 69%, 83%, 11%, and 48%, respectively. The hepatic GOT, LDH, and AP activities increased 56%, 129%, and 51%, respectively. The administration of HgCl2 in PB-pretreated animals was associated with a decrease in the activities of serum GOT and AP (57% and 69%, respectively), while the ICDH activity increased 27%. The hepatic GOT, GPT, and AP increased 58%, 135%, and 77%, respectively, after mercury treatment, whereas LDH and ICDH were inhibited 78% and 29%.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis shows that the conserved cysteine residues of histidinol dehydrogenase are not essential for catalysis.

Histidinol dehydrogenase (HDH) catalyzes two sequential oxidation reactions to produce histidine from histidinol via histidinaldehyde. In HDH proteins so far reported, two Cys residues are conserved. From the results of the studies on Salmonella typhimurium HDH, it has been proposed that one of these two conserved Cys residues is involved in the thiohemiacetal formation at the aldehyde oxidation step [Grubmeyer and Gray (1986) Biochemistry 25, 4778-4784]. To clarify the reaction mechanism, we investigated the role of the conserved Cys residues by site-directed mutagenesis in cabbage HDH. Thus, Cys-112, that corresponds to the catalytic Cys residue of the Salmonella enzyme, and the other conserved one, Cys-149, were replaced with Ala, Ser, or Phe. All the Cys-112 mutant HDHs catalyzed both the alcohol dehydrogenase and aldehyde dehydrogenase reactions, producing 1 mol of L-histidine during the reduction of 2 mol of NAD+, as did the wild type HDH. Site-directed mutagenesis at Cys-149 did not cause significant changes in the catalytic properties, either. These observations, together with the results of detailed comparison of the catalytic properties of mutant HDHs, clearly indicate that neither Cys-112 nor Cys-149 is involved in the reaction, and ruled out the involvement of thiohemiacetal formation in the histidinol dehydrogenase reaction.

Postnatal development, sex-related difference and hormonal regulation of acetohexamide reductase activities in rat liver and kidney.

A variety of patterns of postnatal development were observed for acetohexamide reductase activities in microsomes and cytosols from the liver and kidney of male and female rats. Furthermore, there were pronounced sex-related differences in the activities of liver and kidney microsomes. Prepubertal and neonatal testectomies decreased markedly the activities in liver and kidney microsomes of adult male rats to the female levels. The activities in liver and kidney microsomes decreased by prepubertal testectomy and the activity in kidney microsomes decreased by neonatal testectomy were restored to control levels by testosterone propionate treatment, whereas the activity in liver microsomes decreased by neonatal testectomy was unaffected by the same treatment. Prepubertal or neonatal testectomy had no effect on the activity in liver or kidney cytosol of adult male rats. Prepubertal ovariectomy decreased the activities in liver and kidney cytosols of adult female rats and the decreased activities were restored to control levels by estradiol benzoate treatment. Based on these results, we propose that the postnatal development and sex-related difference of acetohexamide reductase activities in rat liver and kidney can be regulated by sex hormones such as testosterone propionate and estradiol benzoate.

Human chorionic gonadotropin causes an estrogen-mediated induction of rat ovarian carbonyl reductase.

We earlier reported that human chorionic gonadotropin (hCG) stimulates rat ovarian carbonyl reductase (CR) activity and content, and that estrogen enhances the stimulatory effect. The present study was performed to determine the mode of action of the gonadotropin. Cycloheximide (CHX) and actinomycin D (AD) were given to estradiol-pretreated immature rats 6 h before hCG treatment. The enzyme activity was measured with three substrates, and the enzyme content was determined by the method of Western-blot analysis using anti-rat ovarian CR anti-serum as the first antibody. Both protein inhibitors significantly prevented hCG from increasing the enzyme activity and content in estradiol-pretreated ovary. These results indicate that rat ovarian CR is induced by LH via the action of estrogen.