Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a Km for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 microM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca2+, Mg2+, Mn2+, and Ni2+ stimulated calmodulin N-methyltransferase activity but Zn2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory but W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.
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PMID:Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia. 812 67

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (delta hycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (delta hycH) chromatographed with radioactivity from 83Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (delta hypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.
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PMID:Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537. 812 94

Conditions for the overexpression of human wild-type p53 using a baculovirus construct were optimised in insect cells which produced up to 20 mg p53/1 culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double-stranded DNA-cellulose (approximately 58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4 degrees C. The M(r) of extracted p53 both from insect cell lysates and after purification was 54,000 by SDS/PAGE. Isoelectric focusing showed recombinant p53 to be an acidic protein, focusing at pI 6.0 under non-denaturing conditions. Expressed p53 at all stages of purification reacted by immunoblotting with specific p53 monoclonal antibodies, indicating the presence of intact epitopes at the C-terminus, N-terminus and central region of the protein. From ultracentrifugation studies, pure p53 exhibited significant oligomerisation, and sedimented broadly within the 7-12-S region of sucrose gradients. Pure p53 slowly precipitated out of solution at concentrations between 1-6 mg/ml even in the presence of 1% detergent. Using metal affinity chromatography, we have established that pure p53 binds the immobilised divalent ions Zn2+, Ni2+ and Co2+ with high affinity.
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PMID:Biochemical characterisation of purified human wild-type p53 overexpressed in insect cells. 816 7

Oxidative damage induced by NiCl2 to protein has been investigated. We found that nickel induced a dose-dependent increase in the oxidation of bovine serum albumin (BSA) as detected by carbonyl formation in the presence of H2O2 in vitro, as well as producing carbonyl of proteins in intact cultured Chinese hamster ovary (CHO) cells. Other metals capable of producing oxidative damage to BSA in the presence of H2O2 included Cu, Co, Fe, and chromate. However, Cd2+, Hg2+, and Pb2+ were not active even in the presence of H2O2. As an indicator of nickel-induced genotoxic damage, the crosslinking of amino acids to DNA was also examined. Cysteine, histidine, and tyrosine were increased in their association with DNA based upon their persistent binding to DNA following washes with EDTA or SDS. The results suggest that DNA-protein or DNA-amino acids crosslinks induced by nickel may result from interaction of the nickel-oxidized carbonyl group of protein, peptides, and free amino acids.
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PMID:Protein oxidation and amino acid-DNA crosslinking by nickel compounds in intact cultured cells. 820 85

The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HCl and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7% of the original activity. The molecular mass of the purified enzyme was 48,000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8.0-9.8 and 30-35 degrees C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0.1 M-sodium phosphate buffer, but was heat-labile at 50 degrees C in both buffer systems. The activity was inhibited by o-phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.
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PMID:Purification and some properties of proteinase from Pseudomonas fluorescens No. 33. 832 Mar 70

The Bradyrhizobium japonicum heterodimeric nickel-iron hydrogenase efficiently catalyzed H2-ubiquinone-1 oxidoreductase activity at rates up to 47% of the maximal rates obtained using the artificial electron acceptor methylene blue. Gel filtration chromatography and SDS-polyacrylamide gel electrophoresis experiments demonstrated that the purified enzyme was a heterodimer containing only the 65 kDa and 33 kDa subunits. Reduced minus oxidized absorption difference spectra demonstrated the absence of detectable cytochromes. The H2-ubiquinone-1 oxidoreductase activity of both the purified heterodimeric hydrogenase and membranes was significantly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide and antimycin A, inhibitors known to act in the quinone region of electron transport chains. Our results are the first report of H2-ubiquinone oxidoreductase activity by a purified hydrogenase.
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PMID:Hydrogen-ubiquinone oxidoreductase activity by the Bradyrhizobium japonicum membrane-bound hydrogenase. 835 59

Acute stress, such as heat, and some metals, such as arsenite, will induce a specific group of stress proteins referred to as heat-shock proteins. The heat-shock proteins contribute to the survival of cells following a variety of stresses. Similarly, metals such as cadmium and zinc, will increase the levels of metallothionein (MT). The metal-binding protein, MT, has also been found to have a protective role in the cellular response to acute stresses like heavy metals. The purpose of the present study was to examine the production of these proteins in response to metals. Rat hepatocytes were maintained in monolayer culture for 22 hr, and subsequently treated with various concentrations of metals for 4 hr, or incubated at 43.5 degrees C for 15-60 min. Following two washes with fresh media, the cells were labeled with [35S]-methionine (25 microCi/ml) in methionine-free media for 4 hr for determination of heat-shock protein production, or reincubated in fresh media for 20 hr for MT determination. Heat-shock protein production was determined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The autoradiograms were quantified by densitometric scanning. MT was determined by the Cd/hemoglobin affinity assay. Three metals (arsenite, cadmium, and zinc) strongly increased the heat-shock proteins. Whereas arsenite was a much less effective inducer of MT than was cadmium or zinc, arsenite was as effective as the other metals in inducing heat-shock proteins. Nickel was a good inducer of MT; however, it resulted in only a slight increase in the levels of heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Production of metallothionein and heat-shock proteins in response to metals. 836 80

The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure. The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.
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PMID:Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form. 863 80

The human cholecystokinin B (CCKB) receptor was expressed in Sf9 cells by infection with recombinant baculovirus. For immunodetection a c-myc epitope tag (EQKLISEEDL) was fused at the amino-terminus of the CCKB receptor. In a second construct an additional hexa-histidine tag was introduced at the C-terminus of the CCKB receptor to enable employment of metal affinity chromatography. The two receptor constructs were expressed at densities of 6.0 +/- 1.1 pmol/mg protein and 7.2 +/- 1.1 pmol/mg protein, respectively which are 100-200-fold higher compared with the receptor amounts found in natural sources. Saturation of the binding sites with [3H]propionyl-CCK8 revealed Kd values of 4.5 +/- 0.5 nM and 7.8 +/- 0.6 nM for the CCKB receptor without or with histidine tag. In SDS/PAGE and subsequent immunodetection the histidine-tagged CCKB receptor migrated as a 55-kDa band, whereas the CCKB receptor without C-terminal modification revealed apparent molecular masses of 45 kDa and 49 kDa. The differences in the mass values observed for the two constructs suggest that the histidine tag could protect the CCKB receptor against proteolytical degradation from its C-terminus. Furthermore two new photoreactive derivatives of cholecystokinin octapeptide residues 26-33 (CCK8) with high labeling efficiency and specificity for the cholecystokinin receptor subtype B were developed: [3H]BzBz-des-Met28-[p-NH2Bz29]-CCK8 and [3H]BzBz-biotinyl-des-Met28-[p-NH2Bz29]-CCK8. Both contain the p-benzoyl-benzoyl (BzBz) residue at the N-terminus for photoactivation and a p-aminobenzoyl (p-NH2BZ) residue instead of Met28-Gly29 in cholecystokinin. Enzymatic deglycosylation of the CCKB receptor with N-glycosidase F after photoaffinity labeling demonstrated that the CCKB receptor with three potential glycosylation sites was slightly glycosylated, amounting to a molecular mass of about 4 kDa. Using the biotinylated cholecystokinin derivative the photoaffinity-labeled CCKB receptor could be purified 1260-fold by a two-step procedure including affinity chromatography on a streptavidin/avidin agarose matrix. For purification of the native receptor, an improved solubilization protocol for the CCKB receptor using dodecyl beta-D-maltopyranoside was developed. The solubilized CCKB receptors with C-terminal histidine tag retained their ligand binding characteristics after chromatography on a nickel affinity matrix.
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PMID:Photoaffinity labeling of the human brain cholecystokinin receptor overexpressed in insect cells. Solubilization, deglycosylation and purification. 864 24

Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 x g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in a 85% pure preparation with a specific activity of 6.0 U (mg protein)-1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49 degrees C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50-100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.
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PMID:Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans. 866 19


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