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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study was undertaken to investigate glycosylation of porcine enamel proteins secreted in the secretory stage of amelogenesis and to gain insight into functional roles of glycosylated proteins in enamel mineralization. Enamel proteins, isolated from various zones of the secretory enamel, were separated by SDS-PAGE and then transferred on to a nitrocellulose membrane. The transblotted proteins were visualized with either antibodies against porcine amelogenins or various biotin-conjugated lectins. The lectins used were Con-A, GS-II, STA, WGA, s-WGA, GS-I, MPA, VVA, PNA, RCA-I, DBA, SJA, UEA-I, Lotus-A and LPA. The results of the immuno- and lectin blottings revealed that most of the lectins did not bind to porcine amelogenins, while a large number of non-amelogenins having various molecular masses were stained strongly with the conjugated WGA, Con A and MPA lectins. On the basis of the binding specificity with the lectins, porcine non-amelogenins were classified into two groups: WGA (and Con A)-binding moieties at 60-90 kDa (WGA-HMW); and MPA-binding moieties at 13-17 kDa (MPA-LMW). These two groups of non-amelogenins differed distinctly in terms of their localization and stability in the secretory tissue and their adsorption properties onto hydroxyapatite. The WGA-HMW were concentrated in the outer region adjacent to the ameloblasts and disappeared (due to degradation) in the underlying inner secretory enamel. In contrast, the MPA-LMW were found in all zones of the secretory enamel and their quantity remained relatively constant. Histochemical studies using FITC-conjugated WGA and MPA showed that the fluorescence-labelling of WGA was localized in the core region of prism rods, while the fluorescence-labelling of MPA was locally limited at the rim of prism rods or at the prism sheath. In separate adsorption studies, it was found that the WGA-HMW, as well as the intact amelogenins, displayed a high adsorption affinity on to apatite crystals, whereas the MPA-LMW showed only marginal adsorption on to apatitic surfaces. The overall results indicate that part of the heterogeneity found in porcine enamel proteins can be ascribed to variations of carbohydrate moieties attached to non-amelogenins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Localization of glycosylated matrix proteins in secretory porcine enamel and their possible functional roles in enamel mineralization. 133 50

The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.
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PMID:Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin. 133 58

Recently, our laboratory has purified the D1 dopamine receptor 6600 fold to near homogeneity from digitonin solubilized rat striatal membranes using sequential affinity, ion exchange, lectin, and size exclusion chromatographies. The resulting receptor preparations still retained ligand binding activity (-11,000 pmol [3H]SCH 23390 bound per mg/protein) and appeared as a single band at 70-80 kDa on SDS-PAGE. In order to learn more about the sequence and structure of this protein, we recently cloned the gene for a human CNS D1 dopamine receptor. This gene has an open reading frame of 1388 nucleotides and encoded for a protein with a deduced amino acid sequence of 446 residues. When expressed in mammalian cells the cloned D1 receptor had all the ligand binding properties expected for a D1 receptor (SCH 23390 > cis flupenthixol > raclopride and SKF 38393 > apomorphine > dopamine > quinpirole). The cloned D1 receptor was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart, or kidney. These accomplishments now will allow the pursuit of biochemical studies of the receptor protein as well as investigations into structure/function relationship of the receptor using a molecular biological techniques.
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PMID:Location and molecular cloning of D1 dopamine receptor. 136 64

In order to develop a methodology to discriminate fetal bovine serum from other sera with which it might be contaminated, proteins from fetal bovine, newborn calf, colostrum-free newborn, and calf sera were separated by SDS/PAGE and analyzed by silver staining of the gels or lectin blotting, using concanavalin A (Con A), following transfer of the proteins to nitrocellulose. A high molecular weight Con A-reactive glycoprotein of an apparent molecular mass greater than 200,000 daltons was present in newborn, colostrum-free, and calf sera, but absent or at a significantly lower concentration in fetal serum. These two methods accurately and reliably identified contamination of fetal serum with other sera in a series of blind studies on unknown, coded samples. As little as 1% colostrum-free serum in a batch of fetal serum can be detected by either procedure.
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PMID:Discrimination of fetal bovine serum from other sera by silver staining or lectin blotting of SDS/PAGE-separated serum proteins. 136 83

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.
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PMID:A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes. 137 37

A lectin was isolated from the haemolymph of the blowfly larva Calliphora vomitoria. It agglutinated a variety of mammalian erythrocytes with varying specificities and was strongly inhibited by D-galactose and fetuin. The activity was also sensitive to chelators of metal ions, heating above 50 degrees C and proteolytic digestion. SDS-PAGE identified a glycoprotein with an Mr of 32,000 under reducing and nonreducing conditions which resolved to a band at pH 5.4 using isoelectric focusing. Using FPLC gel filtration the activity was isolated in a fraction with an Mr of 130,000. It is suggested that the native form of the molecule is a noncovalently associated tetramer.
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PMID:Purification and characterization of a galactose-specific agglutinin from the haemolymph of the larval stages of the insect Calliphora vomitoria. 137 51

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.
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PMID:Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor. 138 4

A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).
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PMID:Ovarian carcinomas express a sperm acrosomal antigen, defined by monoclonal antibody 9H8. 138 6

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.
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PMID:N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness. 138 2

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.
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PMID:Components and proteolytic processing sites of arylsulfatase B from human placenta. 139 Sep 29


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