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)

The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC-MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS-PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O-glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or beta-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.
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PMID:Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein. 766 86

1. We have purified three P450s from the liver of the phenobarbital (PB)-treated guinea pig in order to evaluate the role of these enzymes in pyrrolizidine alkaloid (PA) metabolism. 2. PB treatment of guinea pig increased the hepatic microsomal conversion of the PA senecionine (SN) to the pyrrolic metabolite (+/-)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), an activation product, and SN N-oxide, a detoxification product by 224 and 70% respectively. 3. Reconstitution of a PB-inducible guinea pig P4502B isoform (M(r) = 57,512 by MALDI-TOF mass spectrometry) in a reconstituted system metabolized SN to DHP and SN N-oxide at rates of 1.98 and 1.45 min-1 respectively. A second purified guinea pig P450, a 2C-type isoform (M(r) = 56,496 by MALDI-TOF mass spectrometry), produced SN N-oxide from SN at the rate of 13.3 min-1 but catalyzed little DHP formation. The third guinea pig P450, an apparent 3A type (M(r) = 54-56,000 by SDS-PAGE), lost its catalytic activity towards SN during the final purification process. 4. Immunoinhibition of microsomal SN metabolism by rabbit antibodies raised against the guinea pig P4502B, 2C and 3A isoforms indicated that the 2B played the most important role (> 70% of the total metabolism) in bioactivation of SN in both the untreated or PB-treated guinea pig, whereas 2C and 3A seemed to exhibit little (around 13%) PA metabolism. P4502B, along with flavin-containing monooxygenase, also contributed to the detoxification of SN in both the untreated (34%) and PB-treated (40%) guinea pig. 5. This study suggests that the putative P4502B form plays the most important role in SN bioactivation in guinea pig.
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PMID:A cytochrome P4502B form is the major bioactivation enzyme for the pyrrolizidine alkaloid senecionine in guinea pig. 855 86

The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457-469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40-50 nm in length and 2.0-2.5 nm in width. A minority of these particles showed a central 'kink'. A number of smaller and generally wider particles (20 nm x 2-4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer-dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 microM and sedimentation velocity data generated a Kd between 1 and 10 microM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.
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PMID:Investigations into the molecular size and shape of tomato extensin. 897 69

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.
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PMID:Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.). 919 23

We have developed HPCE methods for the analysis of sulfomycin (trivial name) and related compounds (code name, crude material = U82127 = I), which is an animal growth promoter derived from a fermentation beer. The crude material, I, isolated from the fermentation consisted of more than 40 components which were not completely separated by conventional HPLC. Thus, as a complementary analysis method, we have optimized HPCE conditions for I using various capillaries including uncoated, coated, and packed using various buffers. The optimized HPCE conditions were obtained with an uncoated fused-silica capillary and a buffer that consisted of 30 mM Tris-tricine, 10 mM SDS, 10 mM NaCl and 20% MeOH, pH 8.0. Using these HPCE conditions, we were able to separate the one main component collected from the HPLC effluent into two or three components. In order to identify the main components of the fermentation product, an off-line HPLC-HPCE-MS analysis for I was performed. From the MALDI-TOF-MS results, the separated components collected from HPCE had different molecular masses. Four lots of I samples having different characteristics were also analyzed by HPCE to investigate lot-to-lot differences in peak profiles. The four lots of I were found to have very similar peak profiles. In this paper, I refers to the crude fermentation product and sulfomycin to the purified, major HPLC component of I.
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PMID:High-performance capillary electrophoresis of a fermentation-derived cyclic peptide analog, animal growth promoter. 934 66

Protein purity estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with scanning densitometry is a critical component in the manufacture of recombinant proteins for commercial diagnostic assays. However, the procedure is time consuming and often difficult to reproduce because commercial dyes that are used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes the use of a rapid and dye-independent SDS polymer-filled capillary gel electrophoresis (CE-SDS) method to estimate protein purity. The CE-SDS method was used for in-process and final purity testing of GB virus-C (GBV-C) fusion proteins produced in E. coli, and was directly compared with the conventional SDS-PAGE method using purified Coomassie blue dye to reduce protein staining anomalies. The CE-SDS method may serve as an alternative or replacement method to the lengthy and tedious SDS-PAGE method. This study also demonstrates that the observed molecular weight of the fusion protein, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides higher accuracy than values estimated by either CE-SDS or SDS-PAGE methods.
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PMID:Capillary electrophoresis for purity estimation and in-process testing of recombinant GB virus-C proteins. 938 14

Blue biliprotein, insecticyanin (INS), has been purified from the hemolymph of the sweet potato hornworm, Agrius convolvuli. The protein was efficiently isolated from the hemolymph of fifth instar larvae using three successive column chromatographic techniques: hydrophobic interaction chromatography, ion exchange chromatography and gel-filtration. The purified INS showed a native molecular weight of approximately 59,000 by gel-filtration. SDS-PAGE revealed a single band with Mr of approximately 26,000. Moreover, the molecular mass of INS was 21,213 by MALDI-TOF/MS. Thus, the native A. convolvuli INS molecule was assumed to be a trimer in solution. The blue coloration of A. convolvuli INS from the hemolymph was attributed to the presence of biliverdin IX gamma, due to the absorbance maxima at 360 and 695 nm, which was non-covalently bound with the apoprotein. Amino acid composition and N-terminal sequence of A. convolvuli INS is similar to M. sexta INS. A. convolvuli INS represents one of the biliverdin-binding proteins in lepidopteran insects.
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PMID:Insecticyanin of Agrius convolvuli: purification and characterization of the biliverdin-binding protein from the larval hemolymph. 945 Mar 89

Human beta-trace protein is a major intrathecally synthesized polypeptide constituent of human cerebrospinal fluid. We have previously shown that this protein is almost quantitatively modified with biantennary complex-type N-linked oligosaccharides which show "brain-type" glycosylation characteristics (Hoffmann,A. et al., J. Neurochem., 63, pp. 2185-2191, 1994). In the present study human beta-trace protein from the cerebrospinal fluid (CSF) of patients with carbohydrate-deficient glycoprotein syndrome (CDGS) due to phosphomannomutase (PMM) deficiency and N-acetyl-glucosaminyltransferase II (GlcNAc-T II) deficiency as well as from control individuals was studied by Western blot analysis. The protein from pooled CSFs was purified by immunoaffinity chromatography. The protein from the five patients with CDGS PMM deficiency showed three protein bands upon SDS-PAGE analysis corresponding to the di-, mono-, and unglycosylated polypeptide forms. Carbohydrate structural analysis of the enzymatically liberated N-glycans was performed applying mapping by HPAEC-PAD, methylation analysis as well as MALD/TOF-MS. Essentially identical oligosaccharide structures were detected in beta-TP from type I patients and control adult pooled CSF. The beta-trace protein from two patients with GlcNAc-T II deficiency showed a single di-N-glycosylated protein band with a significantly lower molecular weight than the di-glycosylated polypeptide from control patients and the beta-trace protein from pooled adult CSF. Beta-TP from GlcNAc-T II deficiency patients shared only three oligosaccharides out of the 13 observed in beta-TP from controls or patients with PMM deficiency. The major oligosaccharide structures of the glycoprotein from patients with GlcNAc-T II deficiency were found to be monoantennary asialo- or monosialylated lactosamine-type chains with proximal fucose and bisecting GlcNAc.
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PMID:Hypoglycosylation of a brain glycoprotein (beta-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency. 945 8

The phosphatase of a psychrophile was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, butyl-Cellulofine, Sephacryl S-100, and Mono-Q columns. The purified enzyme preparation was found to be electrophoretically homogeneous on native- and SDS-PAGE, and its molecular mass was determined to be 38.4 kDa by MALDI-TOF mass spectrometry. Maximal activity was observed at 30 degrees C and pH 6.0. Furthermore, the activity of this enzyme at 0 and 5 degrees C was 27 and 28%, respectively, of that at 30 degrees C. The enzyme was stable in the pH range of 6.0 to 8.0 and up to 20 degrees C. The enzyme was affected by metal ions; the activity was enhanced by Mg2+ and Ca2+ ions, but depressed by Zn2+ ions. Analysis of the amino acid composition indicated that this phosphatase contains no S-S bond, and only a few prolyl residues necessary to retain the rigid structure of a protein molecule. The phosphatase shows typical features of a cold enzyme; high catalytic activity at low temperature and rapid inactivation at an intermediate temperature.
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PMID:Purification and some characteristics of phosphatase of a psychrophile. 953 95

Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS-PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX gamma, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX gamma, whereas BBP-II contains two molecules of biliverdin IX gamma. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.
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PMID:Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. 954 63


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