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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.
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PMID:Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons. 767 15

Preincubation of female rat liver microsomal preparations with [2'-32P]2N3-NADP+ followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14-97 Kd. Labeling of a 26 kD band, the apparent molecular weight of 5 alpha-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP+ (100 microM) completely inhibited the incorporation of [2'-32P]2N3-NADP+ and preserved the enzyme activity, whereas excess NAD+ (100 microM) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained with the detergent-solubilized form of 5 alpha R. Polyethylene glycol (PEG) fractionation of detergent-solubilized preparations of 5 alpha R showed that all the 5 alpha R activity could be recovered in the 6.5% pellet with a 3-4-fold increase in the specific activity. photolysis of this fraction with [2'-32P]2N3-NADP+ resulted in approximately 2-fold increase in 32P labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-32P]2N3-NADP+ indicated that the half-life for photoincorporation and the apparent Kd were 1.0 min and 2 microM, respectively. These results suggest that 2N3-NADP+ is an effective probe of the NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.
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PMID:Photoaffinity labeling of rat liver microsomal steroid 5 alpha-reductase by 2-azido-NADP+. 770 39

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.
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PMID:Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine. 773 2

Integral and peripheral forms of a microsomal retinol dehydrogenase (RoDH) have been distinguished in rat liver through differences in solubility, behavior toward affinity resins, and phase partitioning with Triton X-114. Despite physical differences, polyclonal antibodies raised against integral RoDH recognized peripheral RoDH. No obvious differences were observed in substrate specificity between the two forms. Integral and peripheral RoDH catalyzed retinal synthesis from all-trans-retinol bound to cellular retinol-binding protein, type I (CRBP), with similar Km values of 0.6 and 0.4 microM, respectively. Both also discriminated against CRBP-bound all-trans-3,4-didehydroretinol and against 9-cis-retinol. Phenylarsine oxide inhibited both forms with IC50 values of 5 microM (integral) and 15 microM (peripheral). The more stable peripheral form has been reduced to two major polypeptides that migrate as 34 and 54 kDa bands on SDS-PAGE. The active site of this form has been associated with the 34 kDa polypeptide by covalent binding and inactivation with phenylarsine oxide and by cross-linking to holo-CRBP. Cross-linking required cofactor and was maximum with NADP, consistent with the ordered bisubstrate reaction mechanism of an NADP-supported dehydrogenase. The 34 kDa polypeptide has a subunit molecular weight and other attributes typical of short-chain alcohol dehydrogenases (SCAD) including the highly-conserved SCAD sequence WXLVNNAG, Zn2+ independence; inhibition by carbenoxolone (IC50 = 55 microM), and insensitivity to inhibition by ethanol and 4-methylpyrazole. Tight association between the 34 and 54 kDa polypeptides was demonstrated by their coelution through several columns and the precipitation of RoDH activity with either anti-34 kDa or anti-54 kDa antisera. Because SCAD normally occur as homomultimers, however, the 54 kDa polypeptide is not likely to be a subunit of the peripheral form. This work provides new evidence that the retinol-CRBP "cassette" serves as a substrate for a microsomal RoDH and further characterizes the RoDH.
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PMID:Characterization of a microsomal retinol dehydrogenase: a short-chain alcohol dehydrogenase with integral and peripheral membrane forms that interacts with holo-CRBP (type I). 776 12

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].
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PMID:Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain. 789 62

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.
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PMID:Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase. 806 2

Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE. The enzyme was characterized by Km values of 260 microM for glucose-6-phosphate and 6 microM for NADP and a broad pH optimum between pH 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers. A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.
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PMID:Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.). 818 Jun 21

Cytosolic NADP(+)-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP(+)-ME from tumor cells were identical. Thus, NADP(+)-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue.
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PMID:Characterization of cytosolic malic enzyme in human tumor cells. 818 80

A ferredoxin-NADP(+)-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat.mg protein-1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 microM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chloroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.
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PMID:The purification and properties of ferredoxin-NADP(+)-oxidoreductase from roots of Pisum sativum L. 828 47

Retinal oxidase (retinoic acid synthase) (EC 1.2.3.11) was purified electrophoretically, as a single protein band, from rabbit liver cytosol. The characteristic properties, enzymatic reaction mechanism, substrate specificity and kinetic parameters for retinals and molecular oxygen of the retinal oxidase were investigated. The Km values for all-trans-retinal of the retinal oxidase was the lowest than those for the other retinal derivatives. The retinal oxidase is a metalloflavoenzyme containing 2 FADs as the coenzyme, and 8 irons, 2 molybdenums, 2 disulfide bonds and 8 inorganic sulfurs. Its relative molecular mass was determined to be 270 kDa by gel filtration HPLC on a TSKgel G3000swXL column. Its minimum molecular mass was estimated to be 135 kDa by SDS-PAGE. The optical spectrum of the retinal oxidase showed absorption peaks at 275, 340 and 450 nm, and shoulders at 420 and 473 nm, in the oxidized form. The molecular extinction coefficients of the oxidase at selected wavelengths were determined. Circular dichroism spectra of the retinal oxidase were measured in the ultraviolet and visible regions. These spectra showed positive absorption in the visible region. The amino-acid composition was determined. The activity of the oxidase was not affected by any cofactors, such as NADP+, NAD+, NADPH and NADH, and it did not occur under anaerobic conditions. The oxidase was not inhibited by BOF-4272, a potent inhibitor of xanthine dehydrogenase, or rat anti-xanthine dehydrogenase IgG. Experiments on retinoic acid formation under 18O2 or H2(18)O demonstrated that the oxygen of water was incorporated into retinoic acid by the retinal oxidase, but not molecular oxygen.
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PMID:Characteristic properties of retinal oxidase (retinoic acid synthase) from rabbit hepatocytes. 830 67


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