Effects of dietary powdered green tea and theanine on tumor growth and endogenous hyperlipidemia in hepatoma-bearing rats

The effects of dietary powdered green tea (PGT) and theanine on in vivo hepatoma growth and cancerous hyperlipidemia were investigated in rats that had been implanted with a rat ascites hepatoma cell line of AH109A cells. The hepatoma-bearing rats were fed with a 20% casein diet (20C), 20C containing 2% PGT, or 20C containing 0.1% theanine for 14 days. Dietary PGT significantly and time-dependently reduced the solid tumor volume and weight as did dietary theanine. The hepatoma-induced endogenous hyperlipidemia, which was characterized by rises in the serum cholesterol (hypercholesterolemia) and triglyceride (hypertriglyceridemia) levels, was significantly suppressed by PGT and theanine supplementation. Bile acid excretion into the feces was significantly higher in the PGT- and theanine-fed rats than in the control rats. This inhibition of hypercholesterolemia may have resulted from tumor growth suppression as well as increased excretion of steroids from the body. These results suggest that PGT had both anti-proliferative activity toward hepatoma cells and hypolipidemic activity in the hepatoma bearing rats. They also suggest that theanine was, at least in part, responsible for the PGT actions.     

Increased concentration of CTP synthetase in hepatoma 3924A: immunological evidence

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) was purified 370-fold from rapidly growing rat hepatoma 3924A. A major band was demonstrated by acrylamide gel electrophoresis which corresponded to this enzymic activity. It was estimated that the enzyme was 90% pure. Antibodies were produced in rabbit using this purified hepatoma enzyme. The specificity of the anti-serum was proved by the absence of the reaction between control serum and CTP synthetase. The amount of anti-serum required to inactivate completely the cytosolic CTP synthetase of hepatoma 3924A was 11-fold of that required for normal liver which is in good agreement with the 11-fold increase in CTP synthetase activity in this hepatoma. These results demonstrate that the liver and hepatoma 3924A CTP synthetases were immunologically similar or identical and that the markedly increased enzymic activity in hepatoma 3924A reflected an increase in the enzyme protein amount. These studies provide further evidence that in the neoplastic transformation a reprogramming of gene expression takes place which is manifested in the emergence of increased concentrations of CTP synthetase which should provide selective advantages to cancer cells by increasing the capacity for this rate-limiting step in $\text{de novo}$ CTP biosynthesis.     

Nucleotidase activity in regenerating liver and in clonal strains of hepatoma and pituitary cells in culture.

A nucleotidase of the combined 3'- and 5'-type (nucleotide phosphohydrolase, E.C.3.1.3.31) was present in the cytosol of regenerating rat liver cells, and of rat hepatoma and pituitary cells in culture. The enzyme activity per milligram of cell protein was very similar in regenerating liver and in three of the different cell types. The hepatoma cell strain which showed the slowest growth rate had a three-fold higher basal enzyme activity. After the first days of regenerative growth in rat liver and during early plateau phase of cell growth, there was a 50-120% increase in specific enzyme activity. In the hepatoma cells, the enzyme activities were also compared to the cellular content and synthesis of RNA and DNA. The increase in enzyme activity occurred concomitantly with a reduced incorporation of 3H-thymidine into DNA. The possible physiological role of this nucleotidase in nucleic acid and nucleotide metabolism is discussed.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in minimal deviation hepatoma 7288C. Immunological measurements in hepatoma tissue culture cells.

The mechanism of action of serum lipoproteins and 25-hydroxycholesterol on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells was investigated using antiserum against purified rat liver HMG-CoA reductase (Heller, R. A., and Shrewsbury, M. A. (1976)J. Biol. Chem. 251, 3815-3822). This antiserum cross-reacted with solubilized and membrane-bound HMG-CoA reductase from HTC cells. The enzymes from rat liver and HTC cells appeared antigenically identical. The increase in HMG-CoA reductase activity of HTC cells grown in medium which lacked serum lipoproteins was shown to be due to an increase in immunoprecipitable enzyme. In contrast, the 25-hydroxycholesterol suppression of reductase activity leads to a reduction in the antigenicity of the enzyme rather than a decrease in its number of molecules.     

Light form of Morris hepatoma gamma-glutamyltransferase

Using solubilization with bromelain, the light form of gamma-glutamyltransferase was purified from Morris hepatoma 5123D. Some properties of this enzyme were compared to those of the light form rat kidney GGT. Anthglutin and its isomer inhibit competitively the former enzyme but non-competitively the latter. On zymograms of rat control sera, five GGT fractions were noted, but in sera of rats with hepatoma 5123D also the light form and the increase of GGT activity ain region of fraction II were observed. Only these two enzyme fractions react with antibody anti heavy form of Morris hepatoma GGT.     

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.     

Subcellular localization and isolation of gamma-glutamyltransferase from rat hepatoma cells

Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of gamma-glutamyltransferase ((5-glutamyl-)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2). The activity was exclusively localized to the plasma membrane. Diazotized sulphanilic acid was used as a non-penetrant membrane reagent which inactivates ectoenzymes. With both intact and sonicated cells, only 70-75% inhibition of gamma-glutamyltransferase activity was observed. At least 12% of the total cell complement of gamma-glutamyltransferase activity is highly resistant to inactivation by diazotized sulphanilic acid even after Triton X-100 solubilization. The enzyme was purified from hepatoma cells and its properties compared with enzyme from normal liver. Apart from the striking increase in Vapp there were only minor differences between the enzymes from the two sources. In contrast to the complete abolition of transpeptidase activity of the purified hepatoma enzyme by diazotized sulphanilic acid, the hydrolytic activity of this preparation was only slightly inhibited.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Comparison of the changes in lipid metabolism between hepatoma-bearing and lipopolysaccharide-treated rats

To elucidate the mechanism for hyperlipidemia in the hepatoma-bearing state, changes in some parameters related to the lipid metabolism and serum tumor necrosis factor-alpha (TNF-alpha) level were examined in Donryu rats that had been subcutaneously implanted with an ascites hepatoma cell line of AH109A. These parameters were also examined in rats that had been given a single injection of lipopolysaccharide (LPS), a model for acute infection with TNF-alpha secretion into the blood circulation. The serum triglyceride and total cholesterol (Ch) levels were significantly higher in both the hepatoma-implanted and LPS-injected rats than in normal rats. The level of adipose tissue lipoprotein lipase was decreased by hepatoma implantation and LPS injection, while the hormone-sensitive lipase activity was increased by the same treatments. Fatty acid (FA) oxidation and Ch synthesis were also stimulated by both treatments. The serum TNF-alpha level was noticably elevated by hepatoma implantation and greatly by the LPS injection. This LPS injection increased hepatic FA synthesis. The serum high-density lipoprotein Ch level and hepatic Ch 7alpha-hydroxylase activity were not changed by the LPS injection. Hepatoma implantation led to hyperlipidemia and elevated the serum TNF-alpha level, as did the LPS injection.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Regulation of alkaline phosphatase activity in rat hepatoma cells. Effects of serum proteins, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide

Alkaline phosphatase activity in rat hepatoma cells (R-Y121B) cultured in a monolayer at 0.5% serum was enhanced by serum, bovine serum albumin, casein and gamma-globulin, but ovalbumin, polyvinylpyrrolidone, dexamethasone, insulin and dibutyrylcyclic AMP showed little effect on alkaline phosphatase activity. In addition, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide also increased the enzyme activity, although the incorporation of [14C]leucine into cellular proteins was almost completely inhibited in the presence of these cytotoxic substances. When R-Y121B cell homogenates were incubated at 37 degrees C, alkaline phosphatase activity increased in a pH-dependent manner: the maximal increase was observed at pH 7.1. The magnitudes of the increase differed among cell homogenates and a 4- to 10-fold increase was observed. Alkaline phosphatase in R-Y121B cells was apparently heat-stable, but that in the cells obtained from various treatments was heat labile and the latter activity decreased to less than 50% of the initial activity after 15 min of incubation at 56 degrees C. Alkaline phosphatase in the control and also in the treated cells was more sensitive to L-homoarginine than L-phenylalanine. The Lineweaver-Burk plot showed that the increases in the enzyme activity were accompanied by changes not only in V but also in Km for alkaline phosphatase reaction. Finally, it has been suggested that the increases in alkaline phosphatase activity under various conditions are due to the conversion of the molecule with a low enzyme activity to the molecule with a high enzyme activity in R-Y121B cells.     

Properties of phenylalanine hydroxylase of cultured hepatoma cells.

The kinetic and immunologic properties of phenylalanine hydroxylase of adult rat liver were compared to the properties of the similar enzyme present in cultured H4-II-E-C3 hepatoma cells. The enzymes from the two sources could not be distinguished by the Km values for either phenylalanine or 6,7-dimethyltetrahydropterin. Analysis by double immunodiffusion showed that phenylalanine hydroxylase from the two sources had identical immunologic determinants, but immunotitrations revealed a small but significant difference between the enzyme of the normal adult rat liver and the enzyme of cultured hepatoma cells. The results of double immunodiffusion and immunotitration experiments indicated also that the increased levels of phenylalanine hydroxylase seen in the hepatoma cells grown in the presence of hydrocortisone resulted from the accumulation of enzyme protein, but it could not be decided whether this accumulation resulted from an increased rate of synthesis or decreased rate of degradation.     

Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.     

Biological effects of a nano red elemental selenium.

A novel selenium form, nano red elemental selenium (Nano-Se) was prepared by adding bovine serum albumin to the redox system of selenite and glutathione. Nano-Se has a 7-fold lower acute toxicity than sodium selenite in mice (LD(50) 113 and 15 mg Se/kg body weight respectively). In Se-deficient rat, both Nano-Se and selenite can increase tissue selenium and GPx activity. The biological activities of Nano-Se and selenite were compared in terms of cell proliferation, enzyme induction and protection against free racial-mediated damage in human hepatoma HepG2 cells. Nano-Se and selenite are similarly cell growth inhibited and stimulated synthesis of glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and thioredoxin reductase (TR). When HepG2 cells were co-treated with selenium and glutathione, Nano-Se showed less pro-oxidative effects than selenite, as measured by cell growth. These results demonstrate that Nano-Se has a similar bioavailability in the rat and antioxidant effects on cells.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

The estrogen 2-hydroxylase activity of the gonadotropin-stimulated hypophysectomized immature rat ovary

Using high-performance liquid chromatography and a combination of electrochemical and radiometric flow detection for 2-[14C]hydroxyestradiol, changes in estrogen 2-hydroxylase activity in the microsomal fraction of rat ovarian homogenates were followed. Injection of human chorionic gonadotropin (hCG) at 12-hr intervals to hypophysectomized immature rats stimulated hypertrophy of the theca-interstitial tissue and produced a profound increase in enzyme activity. With the last injection of hCG at 96 hr the peak serum concentration of hCG was reached 12 hr later and then decreased exponentially with a half-time of 13 hr. However, enzyme activity remained elevated for at least 60 hr before beginning to fall. Pregnant mare's serum gonadotropin (PMSG) also produced an increase in activity, which was apparently limited to the thecal-interstitial tissue because freshly removed granulosa cells from the mature follicles had undetectable activity levels. Administration of anti-PMSG antiserum after enzyme activity had been increased resulted in a prompt fall in activity, as did injection of hCG to mimic an ovulatory surge of LH. The results indicate that the thecal-interstitial tissue of the rat ovary has estrogen 2-hydroxylase activity that is dependent upon gonadotropic stimulation for expression.