Inclusion Of Molybdate Research Articles

Peroxomolybdate catalysts in pulp hydrogen peroxide bleaching: Improvement in hexeneuronic acid removal and delignification

We have studied the catalytic effects of peroxomolybdates, i.e., [MoO(O2)2(OH)(H2O)]-, on hydrogen peroxide bleaching on hardwood and softwood pulps in terms of kappa number reduction, hexeneuronic acid (HexA) removal, and delignification. We found that, in the case of hardwood pulp, the decrease of HexA in the peroxomolybdate bleaching stage corresponds to more than 60% of the kappa number reduction obtained in that stage. In the case of pine pulp, inclusion of molybdate in the peroxide stage lowered the kappa number by 4.1. This work also shows that hydrogen peroxide, in the absence of molybdate, did not react to any noticeable extent with neither the lignin model compounds or HexA, under otherwise similar conditions. In order to investigate the oxidation mechanism, lignin model compounds with different side chain structures were reacted with peroxomolybdate. 1H-NMR revealed that lignin model compounds with double bond structure in conjugation with the aromatic ring has an extreme fast speed to react with the peroxomolybdate catalysts systems, presumably through hydroxylation reactions. This mechanism was supported by XPS analysis of the pulp samples, in which the activated hydrogen peroxide was found to act in a similar fashion on surface lignin as well. In conclusion, molybdate activated hydrogen peroxide can be considered a very efficient and selective bleaching agent.

Steroid receptors in breast cancer: Sources of inter-laboratory variation in dextran-charcoal assays

The presently recognized correlations between various clinical parameters and the concentrations of estrogen and progesterone receptors in breast cancer biopsies are largely based on receptor values obtained using the dextran-coated charcoal (DCC) method. This assay method is highly sensitive to slight changes in assay protocol, and differences in assay methodology may account for the wide variation in proportions of receptor positive patients reported by different centers. A survey of various aspects of the assay method that may lead to reproducible, systematic differences in concentrations of receptor levels is presented; and methods of compensating for or correcting these potential differences are discussed. The following aspects are considered: a) constitution of biopsy tissue, b) method of tissue homogenization, c) absorption of ligands to surfaces, d) inclusion of molybdate in the assay buffer, e) composition of the DCC slurry, and f) handling of samples for liquid scintillation counting. Differences in methods used to homogenize tissue in Europe and the U.S.A. may account for differences observed in the correlation of DCC assay results obtained using the recently-introduced monoclonal ER-EIA technique.

The Effect of Including Molybdate in the Eluting Buffer Used for Deae-Cellulose Chromatography on the Quantitation of Glucocorticoid Receptor Transformation

This study analyzes the effect of including molybdate in the elution buffers used in DEAE cellulose chromatography on the fraction of glucocorticoid receptor which elutes as the transformed species. Inclusion of molybdate leads to a significant decrease in the fraction of receptor eluting as transformed; samples which appear to be nearly 50% transformed if eluted in the absence of molybdate were found to be less than 10% transformed when analyzed using a buffer which contained 5 mM molybdate. This decrease was not caused by loss of receptor or a reversion of transformation. DEAE cellulose accelerates receptor transformation. It is concluded that DEAE cellulose should not be used to quantitate transformation unless molybdate is included in all buffers.

Effect of androgen on androgen receptors in cultured human epididymis.

Optimal assay conditions were developed to determine androgen receptor concentrations in samples of human epididymis. Pretreatment of cytosol with mersalyl, as well as the inclusion of molybdate in the homogenization buffers, resulted in a substantial increase in the number of soluble sites detected. A high yield of nuclear and microsomal binding sites was obtained by prolonged incubation (12 h) in 0.6 mol NaCl/l. Organ culture for 6 days resulted in a major loss of androgen-binding sites. In the absence of androgen in the culture media, only 20% of the original sites were found in cultured tissue. Inclusion of dihydrotestosterone (0.1 mumol/l) in the media resulted in samples containing twice as many sites as controls. It is concluded that androgens influence the number of androgen-binding sites in cultured human epididymis in a manner analogous to that described for rat epididymis in vivo.

Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus.

The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.

Electrophoretic analysis of the estrogen receptor. Molybdate stabilization and identification of the classical estrogen receptor.

Conditions are defined which permit analysis of estrogen receptors from the mammalian uterus by polyacrylamide gel electrophoresis, thereby solving a longstanding problem encountered in previous attempts at such analysis, namely the failure of a large portion of the receptor population to enter such gels. A paramount requirement for entry of the estrogen-receptor complex into polyacrylamide gels is its maintenance in an untransformed state which does not form aggregates that are excluded from these gels. Of the multiple estrogen-binding proteins separated, only one (relative mobility of 0.5-0.6) possessed the definitive characteristics of the classical estrogen receptor. The inclusion of molybdate in extraction buffers selectively enhanced receptor recovery and facilitated its separation. Moreover, the estrogen-receptor complex so resolved is separated from other types of estrogen-binding proteins present in the uterine cytosol. These findings show that the molybdate-stabilized estrogen receptor exists in a single discrete form, but otherwise exhibits multiple forms that are probably artifactual. Electrophoresis in discontinuous buffers, but not in a continuous buffer system, promoted aggregate formation. This finding has implications concerning the subunit structure of the untransformed receptor.

Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7

The basis for the glucocorticoid resistance of three (3R7, 3R43 and 4R4) genetically independent mutants derived from the glucocorticoid-sensitive human lymphoid cell line CEM-C7 was examined. Each mutant contained significant, albeit reduced, amounts of steroid binding activity measured in both whole and broken cell assays. These activities were of similar high affinity and specificity to that seen in the sensitive parent, suggesting that the steroid binding portion of the receptors in these mutants was normal. However, nuclear translocation of steroid-receptor (SR) complexes was defective in all three clones. Analysis of SR complex activation by DEAE-cellulose chromatography established that receptors from the mutant clones were extremely labile under conditions which would activate normal SR complexes. Such lability was not exhibited by the unoccupied or occupied but unactivated forms of these receptors, indicating that all three were “activation labile” ( act 1). SR complexes of clones 3R7 and 4R4 were completely protected by the inclusion of molybdate during attempted activation and were classified as act 1:molybdate-sensitive. However, SR complexes of clone 3R43 were unstable during attempted activation, even in the presence of 50 mM molybdate, and were thus classified as act 1:molybdate-resistant. Our results suggest that while the act 1 phenotype may predominate among spontaneously derived glucocorticoid-resistant mutants derived from CEM-C7, this phenotype may be the consequence of at least two different mutations.

Rapid analysis of estrogen receptor heterogeneity by chromatofocusing with high-performance liquid chromatography

Chromatofocusing principles have been utilized to develop a high-performance liquid chromatographic technique for the rapid and routine analysis of steroid receptor heterogeneity. Two anion-exchange columns (SynChropak AX-300 and AX-500) were compared for analytical and preparative chromatofocusing of estrogen receptor components. As many as ten different [125I]iodoestradiol-labeled binding proteins were identified in cytosols prepared from mammary gland and uterus. Estrogen receptors were well separated from other cytosolic proteins and recovery of activity routinely exceeded 90%. Parallel analyses of these cytosols to determine receptor size and shape indicated that HPLC chromatofocusing can be used effectively to study receptor isoforms with Stokes radii ranging from 30 Å to greater than 70 Å. In contrast to isoelectric focusing, this technique is compatible with the inclusion of a commonly used receptor-stabilizing agent, sodium molybdate. Inclusion of molybdate during chromatofocusing of molybdate-stabilized receptor allowed the identification of two acidic receptor species not previously reported.

Molybdate and the measurements of androgen receptors in prostate cancer.

Using the sucrose density gradient technique, we have compared androgen receptor values obtained in the presence and absence of sodium molybdate from cytosols of surgically obtained human prostate cancer tissue. In all nine prostates examined, inclusion of molybdate in the buffers resulted in an increase in the amount of androgen receptor detected, in some cases dramatically. Since accurate androgen receptor assays could be valuable for therapeutic decision making in prostate cancer, we advocate the inclusion of sodium molybdate in assay buffers to guard against the possibility of false negative or artificially low values.

Human Breast Tumor Estrogen Receptor: Effects of Molybdate and Electrophoretic Analyses*

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.

Involvement of Molybdenum and Iron in the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Addition of high levels of sodium molybdate (10-2 m, final concentration) to acid-treated purified bovine milk xanthine oxidase prior to incubation with an extract of the nitrate-induced nitrate reductase Neurospora crassa mutant nit-1 resulted in a significant enhancement of the subsequent in vitro assembly of wild type N. crassa NADPH-nitrate reductase (EC 1.6.6.2). Added molybdate had no effect on the wild type enzyme itself. The enhancement is highly specific for molybdenum, is independent of added phosphate, and also occurs with cell-free preparations of uninduced wild type and with other known acid-treated molybdenum enzymes including chicken liver xanthine dehydrogenase, rabbit liver aldehyde oxidase, bovine sulfite oxidase, and the nitrogenases of Azotobacter and Clostridium. Preincubation at 23° of acid-treated xanthine oxidase preparations resulted in a considerable loss of ability to form nitrate reductase and in a more striking enhancement by subsequently added molybdate. Prior addition of molybdate during the above 23° preincubation prevented in part the loss of the ability of the enzyme to form nitrate reductase, suggesting that the enhancement by molybdate is a restoration effect rather than a stimulatory one. Inclusion of molybdate in the buffer used for homogenization of the uninduced wild type also gave a significant increase in the subsequent in vitro assembly of the enzyme. Various forms and derivatives of molybdenum enhanced to varying degrees the ability of acid-treated xanthine oxidase to form nitrate reductase. Aqueous solutions of several complexes of Mo(V) with cysteine or glutathione under anaerobic conditions failed to substitute for the presumed molybdenum cofactor in the in vitro assembly of nitrate reductase. Sucrose density gradient profiles of NADPH-nitrate reductase formed in vitro by incubation of induced nit-1 extract with a 99Mo-labeled fraction of uninduced wild type (grown on 99MoO4=-containing medium) coincided with that of radioactivity. The addition of 99MoO4= to a cell-free preparation of unlabeled uninduced wild type or to acid-treated xanthine oxidase prior to its incubation with an extract of induced nit-1 gave similar results. Reciprocal experiments using an unlabeled fraction of uninduced wild type and an extract of nit-1 grown and induced in the presence of 99MoO4= showed no incorporation of radioactivity in the in vitro assembled enzyme. Nor was there in vitro exchange or incorporation of 99Mo into the already formed wild type enzyme. Comparable experiments with 55Fe showed that nit-1 and not uninduced wild type or xanthine oxidase is the source of iron in the in vitro assembly of Neurospora NADPH-nitrate reductase.