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)

A gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1. The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M. lacunata. The expressed enzyme from E. coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration. The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5. It had an optimum temperature of 35 degrees C for 5 min of reaction and was stable to up to 35 degrees C for 30 min at pH 7.0. Its molecular weight was estimated to be 80,000 by SDS-PAGE and gel-filtration analyses. It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease. Its amino acid sequence was 38% homologous to that of the E. coli protease II. By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654. The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant.
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PMID:Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme. 762 37

The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34,000 (B-chain)]. Lipase II has a molecular weight of 30,000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6.0-6.5 and a temperature optimum of 35 degrees C, while, for Lipase II these values were pH 6.0 and 40 degrees C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.
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PMID:Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus. 776 29

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

Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61, was purified and partially characterized. The purification was achieved by dialysis against PEG 20,000, butanol extraction and electroendosmotic preparative electrophoresis. The protocol led to a 7843-fold increase in the specific activity, with 3.9% activity recovered. SDS-PAGE of pediocin A resulted in a single 80 kDa protein band. The antimicrobial compound was sensitive to proteolytic enzymes and heat (10 min at 100 degrees C). It exhibited inhibition against species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Enterococcus, Listeria and Clostridium.
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PMID:Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61. 801 91

beta-ketoacyl-ACP synthetase III (KAS III) has been purified from avocado using a six-step purification procedure. The enzyme, which is cerulenin-insensitive and thiolactomycin-sensitive, was assayed using a partial component reaction: acetyl CoA:ACP transacylase (ACAT) activity. KAS III activity is distinguished from ACAT activity on the basis that the former is highly stimulated by the addition of malonyl CoA in the presence of malonyl-CoA:ACP transacylase, and the latter is not. KAS III and ACAT activity have been separated from each other thus providing the first evidence that these two discrete activities exist in higher plants. Both of these enzymes have been implicated in the initial reactions of fatty acid synthesis. KAS III was purified 134-fold using a combination of PEG precipitation, Fast Q, ammonium sulphate precipitation, Phenyl Sepharose and ACP-affinity chromatography. The enzyme requires Triton X-100 for solubility and is highly salt sensitive. The subunit molecular mass of 37 kDa has been identified by SDS-PAGE. The results of gel filtration analysis are consistent with the native enzyme being homodimeric. The native molecular mass of KAS III is 69 kDa and that of ACAT 18.5 kDa. The enzyme has a pH optimum of 7.0-7.5, which is similar to the pH optimum of the ACAT reaction. The Km for acetyl CoA is 12.5 microM and the Km for malonyl-ACP is 14 microM. Both KAS III and ACAT are sensitive to thiolactomycin inhibition. The results are discussed with respect to the potential role of acetyl CoA:ACP transacylase in plants.
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PMID:Acetoacyl-acyl carrier protein synthase from avocado: its purification, characterisation and clear resolution from acetyl CoA:ACP transacylase. 801 68

Monomethoxypolyethylene glycol (mPEG) of average molecular weight 5000 was transformed in a series of synthetic steps to a new activated form of PEG, a stable thiol-protected intermediary, for reaction with cysteine residues in proteins under mild conditions to produce PEG--protein conjugates as possible candidates for therapeutics. The modified protein has PEG polymer molecules attached to the backbone by the newly formed disulfide bonds, which are readily cleaved to regenerate the native protein under mild reducing conditions. The model protein papain, which has seven cysteine residues including a lone cysteine in its active site, was modified through the free available thiol. The resulting PEG--papain was characterized by matrix-assisted laser desorption mass spectrometry, SDS-PAGE, and high performance gel filtration chromatography. The major modified PEG--papain variant was demonstrated to be 5000 Da larger than the unreacted papain.
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PMID:Protected thiol-polyethylene glycol: a new activated polymer for reversible protein modification. 827 13

The authors report on the purification and characterization of mannan- binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan-Sepharose, protein A- and anti-porcine IgM-Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed gamma 1-gamma 2-electrophoretic mobility. The MBP designated pMBP-28 had a molecular mass of 28 kDa when analysed on SDS-PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP-28 revealed an oligomeric protein similar to rodent MBP-A and human MBP but with a predominance of penta- and hexameric molecules. Another protein designated pMBP-27 was composed of peptides of 27 kDa and had an Mr of 300-350 kDa on gel filtration chromatography. Electron microscopy of pMBP-27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N-terminal 26(pMBP-27) and 24(MBP-28) amino acid residues showed 54% and 58% identity with human MBP.pMBP-28 showed a higher degree of sequence similarity to rat and mouse MBP-A (60% identity) than to mouse and rat MBP-C (41-45% identity). Both pMBPs exhibited Ca2+-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was not sequenced.
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PMID:Isolation and characterization of porcine mannan-binding proteins of different size and ultrastructure. 860 63

Selenoprotein Ph, the human analogue of selenoprotein P from rat plasma, was purified from human plasma using Heparin Sepharose chromatography, PEG precipitation, DEAE ion exchange chromatography. RP chromatography, SDS-PAGE and electroelution. SDS-PAGE of the purified protein revealed one broad-selenium containing protein band from 56 to 67 kDa with a selenium maximum at 62 kDa. Using a 7.5% T gel this band was separated into two distinct selenium-containing bands with molecular weights of 61 and 64 kDa.
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PMID:Improved procedure for the purification of selenoprotein Ph from human plasma. 884 59

A simple method is described for the preparation of proteolytically processed forms of beta 2-microglobulin suitable for structural and biological studies. PEG 6000 was added to the serum of healthy individuals to precipitate the C1 complement complex from C1 esterase inhibitor (C1-inh). After dissolving the precipitate containing the C1 complement in Tris-HCl buffer, pH 7.6, efficient conversion of added beta 2-microglobulin to desLys58 beta 2-microglobulin was observed. Addition of a specific carboxypeptidase B inhibitor (Plummers inhibitor) could partly prevent the deletion of Lys-58 from cleaved beta 2-microglobulin, whereby Lys58-cleaved beta 2-microglobulin was obtained. The proteolytically processed forms were subsequently purified by G-75 Sephadex gel filtration followed by chromatofocusing. A yield of 10-40% of proteolytically processed beta 2-microglobulin was obtained. Only one component was seen by SDS-PAGE stained with Coomassie Brilliant Blue.
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PMID:A simple method for the preparation and purification of C1 complement cleaved beta 2-microglobulin from human serum. 923 12

To test the utility of counter-current chromatography in purifying proteins, lactic acid dehydrogenase (LDH) was extracted from a crude bovine heart filtrate using a cross-axis coil planet centrifuge. The purification was performed with several polymer phase systems composed of 16% (w/w) poly(ethylene glycol) (PEG) 1000-12.5% (w/w) potassium phosphate buffers and 4.4% (w/w) PEG 8000-7.0 (w/w) dextran T500 at pH values ranging from 6.5 to 11.0. The best purification was achieved using PEG 1000-potassium phosphate buffer system at pH 7.3 by eluting the upper phase at 1.0 ml/min. Fractions were analyzed by LDH enzymatic activity and sodium dodecyl sulfate slab gel electrophoresis (SDS-PAGE). The LDH was purified directly from bovine heart crude extract within 3 h.
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PMID:Purification of lactic acid dehydrogenase from bovine heart crude extract by counter-current chromatography. 930 Sep 5


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