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

When incubated with activated CoF and normal human serum (as the C source), a portion of the E from most patients with PNH are hemolyzed. In contrast, normal E are completely resistant to this form of C-mediated cytolysis called reactive lysis. This observation implies that normal E express membrane proteins that inhibit reactive lysis. In previous studies, we have shown that when E proteins are subjected to anion exchange chromatography, two peaks of inhibitory activity are observed. We isolated the inhibitor from the first peak and identified it as an 18-kDa protein that we call MIRL (CD59). The purpose of the studies presented herein was to isolate the inhibitory factor (MIRL type II) from the second peak. After anion exchange and gel filtration chromatography, aliquots of the fractions containing MIRL II activity were subjected to preparative SDS-PAGE (nonreducing conditions), and protein was eluted from gel slices electrophoretically. Inhibitory activity was found primarily in two adjacent slices, corresponding to an M(r) range of 84-51 kDa. When MIRL II was analyzed by SDS-PAGE and silver staining, two prominent bands representing proteins with M(r) of 77 and 39 kDa were observed under both reducing and nonreducing conditions. This behavior in SDS-PAGE is characteristic of GP-A, which migrates primarily as a nondisulfide-linked homodimer in equilibrium with a monomeric form. Immunoblotting studies confirmed that MIRL II is GP-A. The protein caused a concentration-dependent inhibition of reactive lysis, with approximately 100 ng producing 50% inhibition. GP-A inhibited reactive lysis by blocking the formation or binding of C5b-7, and this inhibitory activity was immunoprecipitated by monoclonal anti-GP-A. Furthermore, GP-A that was isolated by affinity chromatography also inhibited reactive lysis. These studies demonstrate that the major E sialoglycoprotein functions as an inhibitor of the membrane attack complex of C.
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PMID:Isolation of erythrocyte membrane inhibitor of reactive lysis type II. Identification as glycophorin A. 769 Aug 2

A Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that the Mucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc alpha 1-3GalNAc)-related structures.
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PMID:Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds. 769 47

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

In order to confirm the amino acid sequence predicted from the nucleotide sequence of cDNA and also to elucidate the intracellular localization and molecular evolution, human liver alanine-glyoxylate transaminase 1 (AGT1) was purified and subjected to partial amino acid sequence determination, with special attention to posttranslational modification. The enzyme was purified to homogeneity from the 10,000 x g supernatant of human liver homogenate. The purified enzyme showed only a single protein band at about 43 kDa on SDS-PAGE, indicating that it is a homodimer of two identical subunits, because the native enzyme has a molecular mass of about 80 kDa. Both the amino- and carboxyl-terminal peptides of the enzyme were isolated from a cyanogen bromide digest of the S-carboxyl-methylated protein and subjected to amino acid sequence determination. The alpha-amino group of the amino-terminal peptide was shown to be blocked by an acetyl group. The carboxyl-terminal sequence contained a putative N-glycosylation sequence (-Asn-Ala-Thr-), the only one present in the whole molecule, but this sequence was normally determined, indicating that the enzyme is not N-glycosylated. Purdue et al. [J. Cell Biol. 111, 2341-2351 (1990)] have reported that Pro-11, Gly-170, and Ile-340 in normal human AGT1 were replaced by Leu, Arg, and Met, respectively, in a patient with primary hyperoxaluria type 1. We confirmed that residue-11 was Pro. Both the amino- and carboxyl-terminal sequences of the enzyme showed extensive similarity with those of rat liver mitochondrial serine-pyruvate aminotransferase and the small chain of hydrogenase from a thermophilic unicellular cyanobacterium, Synechococcus PCC 6716.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and amino- and carboxyl-terminal amino acid sequences of alanine-glyoxylate transaminase 1 from human liver. 779 68

A novel membrane-bound serine proteinase has been purified from the microsomal membranes of porcine intestinal mucosa. It was solubilized from the microsomal membrane fraction with 1% sodium deoxycholate, then purified by a series of column chromatographic steps on DE52, butyl-Toyopearl, Bio-Gel P-150, Mono Q, and benzamidine-Sepharose in the presence of 0.02% Lubrol PX. Its molecular mass was estimated to be 50 kDa both by SDS-polyacrylamide gel electrophoresis under non-reducing conditions and by gel filtration, and to be 32 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions, suggesting that the enzyme may exist as a homodimer in which two subunits are linked by disulfide bond(s). It had a pH optimum at around 9 and did not require Ca2+ for activity. It cleaved several peptide 4-methylcoumaryl-7-amide substrates almost exclusively after arginine residues, the best substrate among those tested being t-butyloxycarbonyl-Gln-Ala-Arg-4-methylcoumaryl-7-amide. Various neuropeptides were also cleaved by this enzyme after arginine, mainly between paired basic amino acid residues, Arg-Arg or Arg-Lys. Activity toward protein substrates was scarcely detected. Further, its partial amino acid sequences were highly homologous, but not identical, with those of trypsin-type serine proteinases. These results indicate that the present enzyme is a novel arginine-specific trypsin-like endopeptidase, possibly involved as a processing proteinase in the production of certain gastrointestinal neuropeptides or peptide hormones from their precursors, or their specific degradation.
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PMID:Purification and characterization of a novel membrane-bound arginine-specific serine proteinase from porcine intestinal mucosa. 780 28

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.
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PMID:Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach. 786 30

Centromere protein B (CENP-B) is a common centromere DNA-binding protein among mammalian centromeres. CENP-B possesses the specific DNA binding activity to the 17-base pair sequence dispersed in centromeric repetitive DNA sequences. In the previous study, we have shown that its DNA-binding domain exists within the N-terminal 134 amino acid residues. Here, to clarify the whole domain structure, another functional unit required for CENP-B self-association was examined. Recombinant CENP-B was expressed in Escherichia coli. First, a chemical cross-linking reagent, disuccinimidyl suberate, was used to fix the physical association without losing the DNA-binding activity. The complexes with the same molecular weight as homodimer and trimer were identified after a separation by SDS-polyacrylamide gel electrophoresis. With a series of CENP-B deletion constructs, the area responsible for this oligomer formation was located at the internal region. Second, an electrophoretic mobility shift assay was also used to survey the minimum regions required for the CENP-B self-association. Three separate elements were identified by assaying the capacity to form the additional slow-migrating complex, two in the internal region and one in the C terminus. These results suggest that CENP-B molecules interact with each other at the multiple sites to fold the centromeric DNA repeats into a heterochromatin structure.
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PMID:Functional domain structure of human centromere protein B. Implication of the internal and C-terminal self-association domains in centromeric heterochromatin condensation. 792 83

Characterization of the DNA-binding state of the rat uterine estrogen receptor was examined by UV cross-linking using [32P]5-bromo-2'-deoxyuridine-substituted 25-base pair synthetic oligonucleotide containing a Xenopus vitellogenin A2 estrogen response element (BrdUVRE). After UV irradiation of the receptor-BrdUVRE complexes, they were analyzed by SDS-polyacrylamide gel electrophoresis. In the nuclear estradiol-, ICI164, 384- and 4-hydroxytamoxifen-receptor complexes, the bands corresponding to the mobility of the receptor homodimer were observed. In a molybdate stabilized soluble receptor, we observed the bands with the slower mobility than the receptor homodimers.
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PMID:Characterization of the DNA-binding state of the rat uterine estrogen receptor by UV cross-linking. 795 Oct 63

The human insulin receptor (hIR) is a member of the transmembrane tyrosine kinase receptor family. It is a disulphide-linked homodimer which can be reduced to two insulin-binding monomers by mild reduction of class-I disulphide bonds. The number of disulphide bonds between the alpha- and beta-chains within the monomer or between the monomers in the dimer is not known, although one dimer bond involving hIR Cys-524 has recently been identified [Schaffer and Ljungqvist (1992) Biochem. Biophys. Res. Commun. 189, 650-653]. In the present report hIR Cys-524 was converted into alanine by site-directed mutagenesis and expressed at high levels in Chinese hamster ovary (CHO) cells. The mutant receptor was processed normally and shown to bind insulin normally, with ED50 and KD values not different from those of the wild-type hIR. It was still a disulphide-linked dimer as judged by SDS/PAGE, indicating that there are alpha-alpha-chain disulphide bonds additional to the Cys-524 linkage in the insulin receptor dimer. Insulin-stimulated receptor autophosphorylation and kinase activity of the mutated receptor were both impaired compared with that of the wild-type receptor by 49% and 53% respectively. CHO cells overexpressing the mutant receptor, however, did not show a reduced capacity to stimulate glucose utilization, indicative that the level of receptor expression was sufficient to saturate downstream insulin action. These findings indicate that alpha-alpha disulphides additional to that provided by Cys-524 hold the receptor dimer together and that mutagenesis of Cys-524 reduces the ability of the receptor to signal insulin action subsequent to hormone binding.
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PMID:Cysteine-524 is not the only residue involved in the formation of disulphide-bonded dimers of the insulin receptor. 798 Apr 20


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