Showing papers on "Enzyme assay published in 2001"

Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.

Abstract: The mitochondrial enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3, in the kidney. However, enzyme activity assays indicate that 1α-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1α-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1α-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1α-hydroxylase in both normal and diseased tissues. Specific staining for 1α-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon...

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite.

Abstract: Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation.

Substrate inhibition kinetics for cytochrome P450-catalyzed reactions.

Abstract: Most cytochrome P450 (P450 or CYP)-catalyzed reactions are adequately described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), which are usually determined by a saturation profile of velocity of product formation versus substrate concentration. In turn, these parameters may be used to predict pharmacokinetics. However, some P450 enzymes exhibit atypical or non-Michaelis-Menten kinetics, due largely to substrate inhibition at higher concentrations of substrate. Although the mechanism of substrate inhibition is unknown, ignoring it and truncating the data can lead to erroneous estimates of kinetic parameters. In the present study, 13 P450 marker substrates were examined with 10 recombinant P450 proteins, and 6 were found, to varying degrees, to exhibit substrate inhibition. To understand the nature of the inhibition, a kinetic model was proposed (assuming that two binding sites exist on the enzyme) and used to fit the experimental data. The derived data indicated that 1) the K(I) values (substrate inhibition) were approximately 1.2- to 10-fold greater than the respective K(S) values; 2) both K(S) and K(I) values may be affected by the interaction of the two bound substrates within the enzyme, exhibited by a factor alpha (alpha = 5.1-23.3); and 3) enzyme activity was inhibited markedly (39-97%) at excess concentrations of the substrates (beta = 0.03-0.61). These findings suggest that substrates have access to both the inhibitory site and catalytic site simultaneously (K(I) > K(S)). Furthermore, the two sites, in the presence of substrate, can interact with each other. Therefore, the degree of inhibition of the enzyme is dependent on the concentration of the substrate (usually >K(I)) that sufficiently occupies the inhibitory site.

Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec).

Abstract: The effects of an exogenous enzyme preparation, the application method and feed type on ruminal fermentation and microbial protein synthesis were investigated using the rumen simulation technique (Rusitec). Steam-rolled barley grain and chopped alfalfa hay were sprayed with water (control, C), an enzyme preparation with a predominant xylanase activity (EF), or autoclaved enzyme (AEF) 24 h prior to feeding, or the enzyme was supplied in the buffer infused into the Rusitec (EI). Microbial N incorporation was measured using (15NH4)2SO4 in the buffer. Spent feed bags were pummelled mechanically in buffer to segregate the feed particle-associated (FPA) and feed particle-bound (FPB) bacterial fractions. Enzymes applied to feed reduced neutral-detergent fibre content, and increased the concentration of reducing sugars in barley grain, but not alfalfa hay. Ruminal cellulolytic bacteria were more numerous with EF than with C. Disappearance of DM from barley grain was higher with EF than with C, but alfalfa was unaffected by EF. Treatment EF increased incorporation of 15N into FPA and FPB fractions at 24 and 48 h. In contrast, AEF reduced the 24 h values, relative to C; AEF and C were similar at 48 h. Infused enzyme (EI) did not affect 15N incorporation. Xylanase activity in effluent was increased by EF and EI, compared to C, but not by AEF. Xylanase activity in FPA was higher at 48 h than at 24 h with all treatments; it was higher with EF than C at 24 and 48 h, but was not altered by AEF or EI. Applying enzymes onto feeds before feeding was more effective than dosing directly into the artificial rumen for increasing ruminal fibrolytic activity.

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

Abstract: There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica.

Abstract: The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 μmol min−1 g−1 by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45°C to 60°C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL−1. Myristic acid esters of fructose (22.3 μmol min−1 g−1), α-D-methyl-glucopyranoside (26.9 μmol min−1 g−1) and maltose (1.9 μmol min−1 g−1) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 483–491, 2001.

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

Abstract: An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum Possible existence of a second redox centre is discussed

Influence of supplementary fibrolytic enzymes on the fermentation of corn and grass silages by mixed ruminal microorganisms in vitro.

Abstract: This study was done to determine the effectiveness of supplementary enzymes at increasing the fiber digestion by ruminal microorganisms and to assess whether enzyme activity limits the rate of fiber digestion in ruminal digesta. In vitro comparisons of enzyme activities in two feed enzyme preparations (A and B) with enzyme activities extracted from ruminal fluid indicated that the addition of fibrolytic enzymes at the application rates recommended by the manufacturers would not be expected to increase significantly glycanase and polysaccharidase activities in ruminal fluid. Preparations A and B both increased (P 100 kDa, which is consistent with the cause of the stimulation being enzyme activity. Fibrolytic enzymes from other sources were also able to stimulate gas production: increased rates of gas production were observed in seven out of eight combinations of "cellulase" and corn or grass silage (P 0.05). In contrast, preparations with glucanase activity similar to enzyme A gave at least as great (P < 0.05) an improvement in gas production than enzyme A, irrespective of xylanase activity. It was concluded that enzyme activity, probably a type of endo-(beta-1,4)-glucanase activity, limits the rate of fermentation of corn and grass silage in the rumen. Enzyme supplements of the type used in these experiments are unlikely to possess sufficient activity to overcome this limitation by direct application to ruminal digesta, implying that treatment of the ration prefeeding will be key to harnessing the potential of exogenous fibrolytic enzymes in ruminant nutrition.

Complexation, Stabilization, and UV Photolysis of Extracellular and Surface-Bound Glucosidase and Alkaline Phosphatase: Implications for Biofilm Microbiota.

Abstract: Biofilm-produced and commercially-purified a- and b-glucosidase and alkaline phosphatase were subjected to different spectral portions of natural and artificial light and exposed to various humic substances to elucidate their impact on enzyme activities. Photochemical degradation of all enzymes occurred under different portions of the light spectrum. UVB irradiance produced the greatest overall photochemical degradation of enzymes, with significant rates occurring with UVA and PAR irradiance. The complexation of enzymes with humic substances resulted in inhibition, stabilization, and photochemical protection of the enzyme. Inhibition of enzyme activity occurred via reductions in overall enzyme activity in the presence of humic substances. However, humic-enzyme complexation also resulted in stabilization by restricting enzyme degradation while retaining high activities. Enzymes exposed to natural and artificial light sources had significantly lower reductions in enzyme activities in the presence of humic substances, which indicates that humic-enzyme complexes may protect enzymes from light-induced photochemical degradation. Bacterial surface-bound a- and b-glucosidase activities were significantly reduced in the presence of humic substances. Photosynthetically induced pH changes within biofilm communities can cause large reductions in a- and b-glucosidase activities while enhancing the hydrolytic activity of alkaline phosphatase.

Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase

Abstract: Microbial phytases suitable for food fermentations could be obtained from lactic acid bacteria isolated from natural vegetable fermentations. Phytase activity was evaluated for six lactic acid bacteria cultures. Although the highest activity was found for Lactobacillus plantarum, the phytase activity was very low. Further characterization of the enzyme with phytate-degrading activity showed a molecular weight of 52 kDa and an optimum activity at pH 5.5 and 65 degrees C. Enzyme activity was due to a non-specific acid phosphatase which had a higher hydrolysis rate with monophosphorylated compounds such as acetyl phosphate that could explain the low phytase activity.

Purification and Characterization of Bifunctional Alginate Lyase from Alteromonas sp. Strain No. 272 and Its Action on Saturated Oligomeric Substrates

Abstract: A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).

Purification and characterization of a dimethoate-degrading enzyme of Aspergillus niger ZHY256, isolated from sewage.

Abstract: A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50 degrees C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu(2+). The Michaelis constant (K(m)) and V(max) for dimethoate were 1.25 mM and 292 micromol min(-1) mg of protein(-1), respectively.

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11.

Abstract: The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+ In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate

Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus.

Abstract: Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L.

Abstract: Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be ∼65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 °C. Heat inactivation studies showed temperatures >40 °C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (Km and Vmax values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glut...

A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407

Abstract: Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 degrees C, and was inhibited strongly by Hg2+, Pb2+, Ag+, Fe2+, Mn2+ and Zn2+. The enzyme was stable over a broad pH range 4-8 and below 45 degrees C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg.mL-1 and 1.84 mg.mL-1, respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endo- and exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.

Induction and Regulation of the Carcinogen-Metabolizing Enzyme CYP1A1 by Marijuana Smoke and Δ9-Tetrahydrocannabinol

Abstract: Induction of the carcinogen-metabolizing enzyme cytochrome P4501A1 (CYP1A1) is a key step in the development of tobacco-related cancers. To determine if marijuana smoke activates CYP1A1, a murine hepatoma cell line expressing an inducible CYP1A1 gene (Hepa-1) was exposed in vitro to tar extracts prepared from either tobacco, marijuana, or placebo marijuana cigarettes. Marijuana tar induced higher levels of CYP1A1 messenger RNA (mRNA) than did tobacco tar, yet resulted in much lower CYP1A1 enzyme activity. These differences between marijuana and tobacco were primarily due to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychoactive component of marijuana. Here we show that Delta(9)-THC acts through the aryl hydrocarbon receptor complex to activate transcription of CYP1A1. A 2-microg/ml concentration of Delta(9)-THC produced an average 2.5-fold induction of CYP1A1 mRNA, whereas a 10- microg/ml concentration of Delta(9)-THC produced a 4.3-fold induction. No induction was observed in Hepa-1 mutants lacking functional aryl-hydrocarbon receptor or aryl-hydrocarbon receptor nuclear translocator genes. At the same time, Delta(9)-THC competitively inhibited the CYP1A1 enzyme, reducing its ability to metabolize other substrates. Spiking tobacco tar with Delta(9)-THC resulted in a dose-dependent decrease in the ability to generate CYP1A1 enzyme activity as measured by the ethoxyresorufin-o-deethylase (EROD) assay. This inhibitory effect was confirmed by Michaelis-Menton kinetic analyses using recombinant human CYP1A1 enzyme expressed in insect microsomes. This complex regulation of CYP1A1 by marijuana smoke and the Delta(9)-THC that it contains has implications for the role of marijuana as a cancer risk factor.

Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver

Abstract: Mammalian thioredoxin reductase (TRR; NADPH 2 :oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.