Effect of Oxidized Nucleotide Coenzymes and Mercaptoethanol on Stabilization of Plant Dehydrogenase Activity in Crude Extracts

Activity loss of 6-phosphogluconate, glucose-6-phosphate, and glyceraldehyde-3-phosphate dehydrogenases occurs in crude plant extracts prepared with insoluble polyvinylpyrrolidone (PVP) and a reducing agent, such as mercaptoethanol. Oxidized nucleotide coenzymes (1 mM), in addition to mercaptoethanol (100 mM), in the extraction buffer stabilizes activity of these dehydrogenases in extracts from both woody and herbaceous plants. PVP and mercaptoethanol alone equally stabilize malate dehydrogenase from most species tested. The mercaptoethanol and coenzyme treatment has proven useful in quantification, purification, and characterization of dehydrogenases in physiological investigations.     

The Immobilization Antigen of Paramecium aurelia is a Single Polypeptide Chain

The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethanol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (∼250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.     

Donor substrate specificity and thiol reduction of glutathione disulfide peroxidase.

By isolation of a mixed disulfide product of glutathione and cysteine, glutathione peroxidase was shown to be highly specific for only one donor substrate. Using the coupled assay of NADPH and yeast glutatione reductase, which is highly specific for flutathione disulfide, it was shown that the apparent inhibition of glutathione peroxidase by mercaptoethanol can be described kinetically and that it is competitive with glutathione. Also, when limiting amounts of hydroperoxide were present in the reaction mixture with mercaptoethanol or cysteine, the total amount of glutathione disulfide produced decreased as compared with that in a reaction mixture without mercaptoethanol or cysteine. This finding is consistent with enzymatic formation of mixed disulfides. Data presented suggest that the selenium in glutathione peroxidase was oxidized to a seleninic acid in the absence of glutathione. These results can be explained by a mechanism for glutathione peroxidase wherein the selenium atom is the only atom in the enzyme that undergoes oxidation reduction.     

The Effect of Mercaptoethanol on the Activity of Enzymes of Nitrogen Metabolism in Leaves from Urtica dioica and Spinacia oleracea

The effect of mercaptoethanol at different concentrations on enzyme activity was investigated in leaves from Urtica dioica and Spinacia oleracea. The interference of mercaptoethanol with enzyme activity is dependent on the type of plant, the configuration of the enzyme and the concentration of mercaptoethanol. A stimulation of GDH (glutamate-dehydrogenase) was obtained in both species, while inhibition of GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) was demonstrated in Spinacia. The stimulation of GOT and GPT activity in Urtica was probably due to inhibition of phenol oxidase. Conclusions concerning the effect of mercaptoethanol on NR (nitrate reductase) activity were difficult to draw, since mercaptoethanol itself reduced nitrite and interfered with NR determination in tests in vitro. In Urtica. no activity could be obtained at all with the in vitro method, probably because an inhibitor of NR was liberated during the extraction procedure, Activity of NR could however be obtained in both species when using the in vivo method. Addition of protective agents to the extraction medium has been supposed to influence the protein extractability. In conformity with this increasing amount of fresh matter to the same volume of extraction medium resulted in decreased protein extractability. This led to differences in enzyme activity when expressed on a fresh weight basis but the specific activity remained constant.     

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.     

Energizing plasmalemma of yeasts is necessary for activation of its H+-ATPase by glucose]

ATPase of yeast plasmalemma is known to be activated during incubation of cells or protoplasts with glucose. It has been shown that the level of ATPase activation is sharply decreased after pretreatment of cells or protoplasts with mercaptoethanol, dinitrophenol, gramicidin D, nigericin, or monensin. It is suggested that deenergization of yeast plasmalemma by monensin, nigericin, and mercaptoethanol as uncoupler plays a crucial role in the prevention of in vivo activation of plasma membrane ATPase by glucose. It is concluded that energization of yeast plasmalemma is necessary for activation of ATPase by glucose.     

Antibody Response to Staphylococcus aureus in Rabbits: Sequence of Immunoglobulin Synthesis and Its Correlation with Passive Protection in Mice

Antibody in hyperimmune rabbit antisera specific for Staphylococcus aureus teichoic acid was shown to be associated with the IgM fraction. Treatment of such sera with mercaptoethanol destroyed its activity in passive mouse protection tests, whereas absorption with antirabbit IgG had no effect. Antibody response in normal rabbits immunized by a single or by three daily injections of a killed vaccine of S. aureus was followed by a sensitive passive hemagglutination test. Antibody detected during the "primary" response was completely susceptible to reduction with mercaptoethanol. Most of the antibody detected after a secondary antigenic stimulation at 10 weeks was also susceptible to mercaptoethanol. The antibody titers correlated well with mouse protective activity, and this activity of the serum was also shown to be associated with the IgM fraction.     

Preparation and properties of monomercurated tRNA

Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated tRNA under non-denaturing conditions. The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions. Monomercurated tRNA quantitatively binds to thiopropyl-Sepharose and can be completely desorbed by mercaptoethanol-containing solutions. Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor-dependent polypeptide synthesis in vitro.     

Cysteamine Oxygenase: Possible Involvement of Superoxide Ion in the Catalytic Mechanism

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional properties of the folate transport protein from a methotrexate-resistant subline of Lactobacillus casei.

A methotrexate-resistant subline of Lactobacillus casei has been isolated which transports folate at a reduced rate and contains a binding protein whose affinity for folate (Kd = 280 nM) is considerably lower than that of the corresponding protein of wild-type cells (Kd = 0.6 nM). After the addition of mercaptoethanol, however, this same protein exhibits a high affinity for folate (Kd = 1.2 nM) and transports the substrate at a normal rate. Subsequent removal of mercaptoethanol causes a rapid reversal of the activation process. Binding protein labeled covalently with carbodiimide-activated [3H]folate, solubilized with Triton X-100, and subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate had an apparent molecular weight which was approximately twofold higher than that of the corresponding protein of wild-type cells, but it could be reduced to the parental size (Mr = 20,000) by prior treatment with mercaptoethanol. Purified binding protein also exhibited a similarly elevated molecular weight, and its amino acid composition was indistinguishable from that of the wild-type counterpart, except for the presence of a single cysteine residue. These findings indicate that the mutant binding protein exists in a low-affinity form due to disulfide bridge formation between two homologous protein subunits and that cleavage of this bond by mercaptoethanol generates the high-affinity state. The rapid and specific interconversion of these binding forms suggests further that the high-affinity form of the binding protein also resides in the membrane as a dimer, held together by noncovalent interactions.     

Reversible alkylation of an active site methionine residue in dehydroquinase

Iodoacetic acid inactivates dehydroquinase by simultaneously alkylating 2 methionine residues (Met-23 and Met-205), presumed to be active site residues (described in Kleanthous, C., Campbell, D. G., and Coggins, J. R. (1990) J. Biol. Chem. 265, 10929-10934). Although both sites are carboxymethylated to the same degree in the inactivated enzyme, the modification of Met-205 may be reversed by treatment with mercaptoethanol at alkaline pH, as shown by the stoichiometric loss of label from this site. This, in turn, leads to partial reactivation of the inactive enzyme. Alkylation of Met-23 is not reversible under these conditions. The chemistry of the cleavage reaction at Met-205 was investigated by isolating the cleavage product which was identified by mass spectrometry as the ammonium salt of 2-hydroxyethyl thioacetate. This result is consistent with nucleophilic attack by the thiolate anion of mercaptoethanol on the alpha-carbon of the carboxymethyl moiety, which restores the side chain of the methionine residue (Met-205) and liberates 2-hydroxyethyl thioacetate. The differential reactivity of the 2 carboxymethylated methionine residues toward mercaptoethanol is likely to be a reflection of their different microenvironments in the folded protein. This assertion is borne out by unfolding experiments which indicate that neither of the carboxymethylated methionine residues in dicarboxymethylated dehydroquinase is susceptible to mercaptoethanol cleavage if the protein is first denatured by either guanidine hydrochloride or urea. Furthermore, this denatured material refolds after removal of denaturant to yield protein with reactivation properties similar to untreated, dicarboxymethylated enzyme.     

Spectroscopic properties of tetrapyrroles on denatured biliproteins

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.     

Effect of 2-mercaptoethanol on the individual periods of the mitotic cycle

Dynamics of the mitotic cycle of the KEPV cells being on different interphase stages at the start of a 20 hour 2-mercaptoethanol (0.001 M) treatment has been studied during the treatment and for 11 hours after washing out the agent. The KEPV cells affected by mercaptoethanol during the interphase (G1, S, G2) were shown to continue their passage through the cycle to enter mitosis, but part of the cells of the S period and of the first half of the G2 period were arrested in the interphase. In the presence of mercaptoethanol, mitotic cells reach the metaphase stage, and their further behaviour depends on the duration of the treatment. For the first 8 hours of treatment, a phase of "unstable block" exists for cells that were in S and G2 periods at the beginning of treatment, while other cells are transformed into K-metaphases. 8 hours later a phase of "stable block" occurs and all the normal metaphases are transformed into K-metaphases. After washing out the culture from mercaptoethanol the cells are ejected from the block in K-metaphase. The transformation from K-metaphase into the normal metaphase is realised in the course of this process. The cells which were in S and G2 periods at the beginning of the treatment are ejected from the block simultaneously after washing, while the cells of the G1 period--with a small delay. After washing out mercaptoethanol the cells that were in the interphase (G1, S, G2) at the beginning of the treatment are capable of producing both multipolar mitoses and mitoses without cytotomy.     

Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD+-15-hydroxyprostaglandin in dehydrogenase.

The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of 3H-PGE1 to 15-keto-3H-PGE1. Mercaptoethanol reacted with 15-keto-PGE1 to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE1. The implication of the use of ME in radiometric assays is discussed.     

Effects of sulfhydryl compounds on the inhibition of erythrocyte membrane Na+(-)K+ ATPase by ozone

Exposure of human erythrocyte membranes to ozone (5 mumol/10 min) resulted in the inhibition of erythrocyte membrane Na+(-)K+ ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0-1.0 mM) of dithiotrethiol or mercaptoethanol Na+(-)K+ ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+(-)K+ ATPase value. The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme protein.     

Escherichia coli 987P pilus: purification and partial characterization

The Escherichia coli somatic pilus, 987P, has been purified after removal by homogenization from a 987P+ enterotoxigenic E. coli. Cell-free pili were precipitated by the addition of MgCl2, collected, and dissolved in MgCl2-free buffer. Five cycles of precipitation and dissolving resulted in a preparation of 987P that was judged to be homogeneous based on electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the electron microscope, 987P was rod shaped, having a diameter of 7 nm and an apparent axial hole. Cells and membrane vesicles were not observed in the purified pilus preparation. Electrophoresis of 987P through sodium dodecyl sulfate-polyacrylamide gels resulted in a single band when the sample was denatured in the absence of mercaptoethanol and in two bands when the sample was denatured in the presence of mercaptoethanol. The calculated molecular weight of 987 was variable, depending upon the polyacrylamide concentration and whether mercaptoethanol was included in the denaturing solution. Chemically, 987P is composed primarily of protein but also contains an unidentified amino sugar. The amino terminal amino acid of 987P is alanine and its isoelectric point is pH 3.7. 987P possesses no detectable hemagglutinating activity.     

Specific inhibition of sclerotium formation by 2-mercaptoethanol and related sulfhydryl compounds in Sclerotium rolfsii.

Sclerotium formation in Sclerotium rolfsii was completely inhibited by 2-mercaptoethanol at a concentration of 2-4 mM without any adverse effect on mycelial growth. Concentrations lower than 2 mM had no effect on mycelial growth and sclerotium formation, whereas both were inhibited at concentrations higher than 4 mM. Complete inhibition of sclerotium formation with no effect on mycelial growth was also obtained by propyl mercaptan, 1-butyl mercaptan and 2-butyl mercaptan at a concentration of 0.10 mM. Sclerotium formation was also inhibited by benzyl mercaptan and thioglycolic acid at 0.15 mM and 2-4 mM concentration respectively, whereas it was only partially inhibited by L-cysteine and glutathione at 20 mM. Mycelium grown for 21 days in nutrient medium supplemented with mercaptoethanol at a concentration of 3 mM, when transferred into fresh medium without the chemical, grew normally and produced abundant mature sclerotia. Mercaptoethanol inhibited the initiation as well as the further development of young, unpigmented sclerotia. The mechanism of sclerotium formation was arrested completely when mercaptoethanol was added to the growth medium at any time between inoculation and the appearance of sclerotia of the "development" stage. It is suggested that the specific inhibitory action of mercaptoethanol could be used to study the mechanism of sclerotium formation     

The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria.

Electron-transferring flavoprotein (ETF) and acyl dehydrogenases of pig liver mitochondria have been isolated in good yield by a new procedure. ETF and general acyl dehydrogenase appear homogenous, are free of reciprocal contamination, react with neither pyridine nucleotides not cytochrome c, and are completely dependent upon each other for reduction of dichlorophenol indophenol by acyl-CaA substrates. The properties of the present preparation (some of which differ significantly from those previously described) are presented. Sedimentation of ETF in 0.02 M KP-i yields a M-r for the native ETF of 58,00 plus or minus 3,000, whereas sedimentation of reduced and alkylated ETF in guanidine HCl yields a M-r of 26,000. Electrophoresis on sodium dodecyl sulfate gels in the presence or absence of mercaptoethanol gives a M-r of about 27,000 and flavin analysis gives a minimum molecular weight of about the same figure. Thus, ETF appears to contain one flavin (at least 90% FAD, by chromatographic and fluorescence characteristics) per 26,000 M-r, and therefore may be composed of two subunits with one flavin each. Sodium dodecyl sulfate gel electrophoresis of general acyl dehydrogenase in the absence of mercaptoethanol gives a band corresponding to a M-r of 84,000; in the presence of mercaptoethanol a band corresponding to a M-r of 42,000 is found. The minimum molecular weight based on flavin content is 40,500. These data considered in conjunction with previous reports from other laboratories, suggest a structure of four subunits per mol with one flavin per subunit..     

Comparison of molecular properties of rat plasma and erythrocyte selenium-dependent glutathion peroxidase

The molecular structure of plasma and erythrocyte selenium-dependent glutatione peroxidase (GSH-Px) was studied in rats drinking water containing [⁷⁵Se]selenious acid, 1.3 mg Se/L. Substantial differences were found using three-step fractionation, including gel filtration of crude plasma and erythrocyte lysate, gel filtration of⁷⁵Se-GSH-Px treated by mercaptoethanol, and SDS-electrophoresis. Native plasma⁷⁵Se-GSH-Px, which exhibited a molecular weight (M _r) of approx 700,000, could be destroyed by mercaptoethanol action, resulting in disintegration of enzyme into several different⁷⁵Se-protein fragments and release of part of low-mol-wt⁷⁵Se. Native erythrocyte⁷⁵Se-GSH-PxM _r, value was found to be 113,000; two⁷⁵Se-protein fragments arose after mercaptoethanol treatment without⁷⁵Se release from the enzyme. The⁷⁵Se-subunits of 22,500 and 21,900 were isolated from plasma and erythrocyte⁷⁵Se-GSH-Px, respectively. Another minor⁷⁵Se-GSH-Px was identified in erythrocyte lysate (M _r, 214,000, subunit 22,100), which was considered to be a dimer of the above-mentioned erythrocyte enzyme. It can be assumed, based on these data, that native plasma GSH-Px, in contrast to erythrocyte enzyme, represents a high-molecular wt complex composed of several tetramers linked with S—S bonds. A certain part of selenium present in this complex is probably not selenocysteine and may be released with the mercaptoethanol treatment.