Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein

SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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“…Fractions of 0.5 ml were collected and assayed for protein concentration (29), refractive index (% sucrose), and ATPase and PNPPase activities.…”

Section: Sucrose Density Gradient Centrifugation Of Membrane-bound Namentioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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“…The mixture was incubated for 2 h at 37°C, 30 p1 33% acetic acid was then added and 300 p1 of the neutralized solution was taken for scintillation counting. A further 50 pl was removed for measurement of protein concentration by the method of Read & Northcote (1981).…”

Section: Glucose Uptakementioning

confidence: 99%

Anti-tumour cytotoxin produced by human monocytes: Studies on its mode of action

Matthews¹

1983

Br J Cancer

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Summary Human monocytes can be induced to synthesize a cytotoxin which affects certain tumour cell lines. The interaction of monocyte cytotoxin with a susceptible cell line (L929) has been studied to obtain clues to the mode of action of the cytotoxin. The cytotoxin acts directly on the cells rather than on the culture medium and is cytotoxic at higher concentrations and cytostatic at lower concentrations. First signs of cell damage appear about 20 h after contact with the cytotoxin which must be present throughout this period. The cytotoxin probably acts on the cell surface and is more effective at 40°C than at 37°C. For a given amount of cytotoxin the effects are inversely proportional to the target cell concentration. Treatment of the cytotoxin with phenanthroline inhibits cytotoxicity while treatment of the target cells with actinomycin D, but not cycloheximide or puromycin, enhances cytotoxicity. After 24h cytotoxin treatment the target cells exhibit reduced respiration rate but enhanced glycolysis and glucose uptake suggesting mitochondrial dysfunction. A possible interpretation of these data is that the monocyte cytotoxin is a metalloenzyme which inactivates a cell surface receptor for a nutrient essential for mitochondrial function.A number of macrophage products with antitumour properties have been defined using in vitro assays. These products include arginase (Currie, 1978), cytolytic factor (Adams et al., 1980), tumour necrosis factor (Miinnel et al., 1980; Matthews, 1978, 198 la), human monocyte cytotoxin (Matthews, 1981b) and cytostatic factors CFI and CFII from human monocytes (Nissen-Meyer & Hammerstrqm, 1982). These factors may act separately or in concert (Adams et al., 1981;Nissen-Meyer and Hammerstr0m, 1982;Matthews, 1983).The human monocyte cytotoxin is apparently specific for certain tumour cell lines, it lacks species specificity and has a mol wt of -34,000 and slow electrophoretic mobility (Matthews, 1981b plastic adherence, were cultured overnight in Eagles minimum essential medium with 10% foetal calf serum (MEM/FCS) and 10 ugml-' endotoxin (Matthews, 1981b). A cytotoxin-enriched fraction was prepared from the monocyte supernatant by ion-exchange chromatography with CM-Sepharose (Matthews, 1983). Cytotoxin assay The mouse L929 tumour cell line was used as the target cells. Seventy-five pl amounts of target cell suspension (10 ml'1 in MEM/FCS) were pipetted into 96 well microtitre trays and incubated for at least 4h to allow the cells to adhere. Cytotoxin preparations were added in 75pl amounts, usually at 3 dilutions and with 3 or 4 replicates/dilution. After incubation at 37°C for 2-3 days the supernatant containing the dead cells was discarded and the adherent viable cells were fixed for 5min with 5% formaldehyde and stained with crystal violet. After drying, 100p1 33% acetic acid was added to each well to dissolve and evenly spread the dye. The amount of dye bound is proportional to the number of viable cells and was quantitated photometrically using a Titertek Multiskan photometer...

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“…Protein concentration was determined by a modified Bradford method (27). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein samples was done by the procedure of Laemmli (21), and gels were stained with Coomassie blue.…”

mentioning

confidence: 99%

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

et al. 1993

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The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:NM-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme 13-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of 1-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, lucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state. The enzyme was stringently specific with regard to its substrate and cofactor, L-lysine and flavin adenine dinucleotide, respectively. NADPH appeared to be the preferred electron donor used by IucD439 in the N hydroxylation process.

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Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein

Cited by 1,355 publications

References 19 publications

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Anti-tumour cytotoxin produced by human monocytes: Studies on its mode of action

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

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