Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C

The tetrazolium salt 3(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) is reduced to formazan by the succinate dehydrogenase system of active mitochondria, and hence, specifically used to assay for the viable cells, such as measurement of cell proliferation, cytotoxicity, and cell number. However, in the present study we have shown that some component specifically present in M199 but not in RPMI 1640 media can reduce MTT to formazan in the absence of a living system. Further study revealed that ascorbic acid reduced MTT to formazan, which was profoundly increased by a very small amount of retinol, whereas retinol alone had no effect. Oxidation of ascorbic acid by H₂O₂ destroyed its ability to reduce MTT. The rate of MTT reduction was directly proportional to the concentration of MTT in the absence of retinol, but approached a zero‐order state beyond a certain concentration of MTT in the presence of retinol. Furthermore, retinol remained unchanged after the completion of the reaction. Taken together, these results showed that retinol acts as a reductase that catalyzes the reduction of MTT to formazan using ascorbic acid as the cosubstrate (electron donor). J. Cell. Biochem. 80:133–138, 2000. © 2000 Wiley‐Liss, Inc.     

Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid beta peptides (Abeta) and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol), we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Abeta. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.     

Structure of a novel enzyme that catalyzes acyl transfer to alcohols in aqueous conditions

The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.     

Purification and properties of L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the formation of beta-alanine in Escherichia coli.

L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the production of beta-alanine, has been purified to apparent homogeneity from Escherichia coli. The properties of the enzyme are: (a) pH optimum of 6.8 to 7.5, (b) temperature optimum of 55 degrees C, (c) Km for L-aspartate of 0.16 mM, and (d) molecular weight of 58,000. The activity of the enzyme is inhibited by reagents (hydroxylamine, phenylhydrazine, and sodium borohydride) that react with carbonyl groups, but no pyridoxal phosphate is present. The compound containing the carbonyl group has been identified as covalently bound pyruvate. Approximately 1 mol of pyruvate was found/mol of enzyme. That the enzyme has a biosynthetic function rather than a catabolic role is indicated by the observations that a mutant (designated as E. coli 99-2) which requires either beta-alanine or pantothenic acid for growth contains only trace amounts of enzyme activity, whereas it is present in substantial amounts in the parent strain (E. coli W) and in a spontaneous revertant of the mutant.     

The competition plot: A kinetic method to assess whether an enzyme that catalyzes multiple reactions does so at a unique site

Enzymes often act on more than one substrate, and the question then arises as to whether this can be attributed to the existence of two different enzymes that have not been separated or, more interesting, to the presence of two different active sites in the same enzyme. The competition plot is a kinetic method that allows us to test with little experimentation whether the two reactions occur at the same site or at different sites. It consists of making mixtures of the two substrates and plotting the total rate against a parameter p that defines the concentrations of the two substrates in terms of reference concentrations chosen to give the same rates at p = 0 and p = 1, i.e., when only one of the substrates is present. With a slight modification of the equations it can also be applied to enzymes that deviate from Michaelis-Menten kinetics. If the two substrates react at the same site, the competition plot gives a horizontal straight line; i.e., the total rate is independent of p. In contrast, if the two reactions occur at two separate and independent sites a curve with a maximum is obtained; separate reactions with cross-inhibition generate curves with either maxima or minima according to whether the Michaelis constants of the two substrates are smaller or larger than their inhibition constants in the other reactions. Strategies to avoid ambiguous results and to improve the sensitivity of the plot are described. A practical example is given to facilitate the experimental protocol for this plot.     

The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C.

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.     

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.     

Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis

The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups of acetyl-CoA, then equilibrate the carbonyl with CO in the solution and re-form acetyl-CoA. CoA is not necessary for the exchange and, in fact, inhibits the reaction. These studies support the view that CO dehydrogenase is the condensing enzyme that forms acetyl-CoA from its component parts. Carbon dioxide also exchanges with the C-1 of acetyl-CoA, but at a much lower rate than does CO. At 50 degrees C and pH 5.3, the optimal pH, the turnover number is 70 mol of CO exchanged per min/mol of enzyme. Low potential electron carriers are stimulatory. The Km app for stimulation by ferredoxin is 50-fold less than the value for flavodoxin. Neither ATP or Pi stimulate the exchange. The EPR spectrum of the CO-reacted enzyme is markedly changed by binding of CoA or acetyl-CoA. Arginine residues of the CO dehydrogenase appear to be involved in the active site, possibly by binding acetyl-CoA. Mersalyl acid, methyl iodide, 5,5-dithiobis-(2-nitrobenzoate), and sodium dithionite inhibit the exchange reaction. A scheme is presented to account for the role of CO dehydrogenase in the exchange reaction and in the synthesis of acetate.     

Relationship of MTT reduction to stimulants of muscle metabolism

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.     

TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules

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Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

Targeted disruption of tyrosylprotein sulfotransferase-2, an enzyme that catalyzes post-translational protein tyrosine O-sulfation, causes male infertility

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.     

Old Yellow Enzyme from Candida macedoniensis catalyzes the stereospecific reduction of the C=C bond of ketoisophorone

Microorganisms were screened for ones that reduced 3,5,5-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone; KIP), and several strains were found to produce (6R)-2,2,6-trimethylcyclohexane-1,4-dione (levodione). The enzyme catalyzing the reduction of the C=C bond of KIP to yield (6R)-levodione was isolated from Candida macedoniensis AKU4588. The results of primary structural analysis and its enzymatic properties suggested that the enzyme might be an Old Yellow Enzyme family protein.     

Vitamin C partially reversed some biochemical changes produced by vitamin E deficiency

Feeding a basal diet free of vitamins E and C to weanling male rats for 8 months resulted in biochemical changes characteristic of vitamin E deficiency. These included increased liver thiobarbituric acid values; decreased blood GSH levels, plasma vitamin E levels, and glutathione peroxidase activities; and increased activities of plasma pyruvate kinase, glutamic-oxaloacetic transaminase, creatine kinase, lactic dehydrogenase, and malic dehydrogenase. Tube-feeding vitamin C for 21 days resulted in partial reversal effects on the above parameters except activities of glutathione peroxidase, lactic dehydrogenase, and malic dehydrogenase. The results suggest that vitamin C may spare in part the metabolism of vitamin E through its antioxidant property.     

Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide.

Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.     

Recombinant type A rat 75-kDa alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an alpha-hydroxyglycine intermediate

The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine alpha-amidating enzyme (alpha-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A alpha-AE secrete the expected 75-kDa enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-Tyr-Val-Gly and dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A alpha-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-alpha-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second enzyme to convert peptidyl-alpha-hydroxyglycine to peptidyl-NH2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.     

Molybdenum nitrogenase catalyzes the reduction and coupling of CO to form hydrocarbons

The molybdenum-dependent nitrogenase catalyzes the multi-electron reduction of protons and N(2) to yield H(2) and 2NH(3). It also catalyzes the reduction of a number of non-physiological doubly and triply bonded small molecules (e.g. C(2)H(2), N(2)O). Carbon monoxide (CO) is not reduced by the wild-type molybdenum nitrogenase but instead inhibits the reduction of all substrates catalyzed by nitrogenase except protons. Here, we report that when the nitrogenase MoFe protein α-Val(70) residue is substituted by alanine or glycine, the resulting variant proteins will catalyze the reduction and coupling of CO to form methane (CH(4)), ethane (C(2)H(6)), ethylene (C(2)H(4)), propene (C(3)H(6)), and propane (C(3)H(8)). The rates and ratios of hydrocarbon production from CO can be adjusted by changing the flux of electrons through nitrogenase, by substitution of other amino acids located near the FeMo-cofactor, or by changing the partial pressure of CO. Increasing the partial pressure of CO shifted the product ratio in favor of the longer chain alkanes and alkenes. The implications of these findings in understanding the nitrogenase mechanism and the relationship to Fischer-Tropsch production of hydrocarbons from CO are discussed.