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)

Spectrophotometric and gas-liquid chromatographic analyses on the carbohydrate moiety of tryptic erythrocyte glycopeptides from persons with Tn-syndrome reveal a selective lowering of the galactose and sialic acid content, the degree being dependent on the percentage of polyagglutinable cells. Alkaline borohydride specifically releases N-acetylgalactosaminitol, and the amount is correlated to the percentage of pathological acetylgalactosaminitol, and the amount is correlated to the percentage of pathological erythrocytes. It is concluded that the alkali-labile carbohydrate chains of Tn-polyagglutinable red cells solely consist of N-acetylgalactosamine linked to serine or threonine. Experiments with heterophile agglutinins whose specificity is known are in line with the above-mentioned results. As judged from SDS-polyacrylamide gel electrophoresis the three major membrane glycoproteins are affected to a different extent by the defect.
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PMID:Molecular basis of Tn-polyagglutinability. 117

A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.
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PMID:A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors. 128 Jan 10

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.
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PMID:Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein. 131 95

A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.
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PMID:An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7. 131 73

pp59v-rel exists in association with specific cellular proteins within lymphoid cells transformed by reticuloendotheliosis virus (REV-T). These include the cellular rel homolog (p75c-rel) as well as a 40-kDa avian homolog to I kappa B. The brief exposure of REV-T-transformed lymphoid cells to micromolar concentrations of ZnSO4 induces profound alterations within these protein complexes. Most of the constituents of the rel protein complexes (to include pp59v-rel, p75c-rel, and p115) translocate from the cytosol to the nucleus. This system has been used to characterize the molecular events that accompany the activation of rel protein complexes. The level of phosphorylation increases on three proteins within these complexes: pp59v-rel, p75-c-rel, and pp40. The degree of phosphorylation on pp59v-rel is such that its relative mass increases 3 to 6 kDa when resolved by SDS-polyacrylamide gel electrophoresis. pp59v-rel is phosphorylated on serine and threonine residues predominantly within a single domain of 17.5 kDa. Similarly, p75c-rel exhibits a corresponding increase in its relative mass with increased phosphorylation. The increased phosphorylation of pp40 is accompanied by its dissociation from the cytosolic rel protein complexes. These observations draw parallels with the induction of the NF-kappa B trans-activating factor.
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PMID:Alterations within pp59v-rel-containing protein complexes following the stimulation of REV-T-transformed lymphoid cells with zinc. 131 81

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.
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PMID:Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity. 132 Oct 48

Nerve growth factor (NGF) binds to two structurally unrelated transmembrane proteins on the surface of PC-12 cells, a 75-kDa glycoprotein with a short cytoplasmic sequence, and the trk protooncogene (pp140c-trk), a protein tyrosine kinase activated by NGF. Immediately after binding to cells, NGF induces changes in serine/threonine phosphorylation of several proteins. We have explored the relative roles of these two NGF binding proteins in mediating the activation of two intracellular kinases that may be responsible for some of these phosphorylations. The raf-1 protooncogene is a serine/threonine kinase activated by several growth factors and oncogenic proteins. Treatment of PC-12 cells with NGF increases the serine and threonine phosphorylation of raf-1 in an anti-raf-1 immunoprecipitate kinase assay. This increased phosphorylation observed in vitro is dose-dependent and transient and is accompanied by the NGF-dependent shift in the mobility of immunoblotted raf-1 on SDS sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an effect thought to reflect phosphorylation. NGF-dependent activation of raf-1 is not dependent on protein kinase C, since prolonged exposure to phorbol esters under conditions that cause down-regulation of cellular protein kinase C activity has no effect on the NGF response. Expression of pp140c-trk in 3T3 fibroblasts (3T3-c-trk), as evidenced by cross-linking of 125I-NGF to the 140-kDa protein, permits the NGF-dependent activation of raf-1 kinase, detected in the immunoprecipitate kinase assay, anti-raf immunoblot shift on gel electrophoresis, and incorporation of [32P]orthophosphate into the raf-1 protein. The concentration dependence of raf-1 activation is identical in 3T3-c-trk and PC-12 cells, despite the absence of the 75-kDa NGF binding protein in 3T3-c-trk cells. NGF is without effect in untransfected 3T3 cells or in Chinese hamster ovary cells overexpressing p75, although raf-1 is present in these cells. Similarly, the NGF-dependent activation of mitogen-activated protein (MAP) kinase is detected in 3T3-c-trk cells, but not in untransfected 3T3 or Chinese hamster ovary cells overexpressing p75. As described for raf-1 activation, the NGF dose responses for MAP kinase activation in 3T3-c-trk and PC-12 cells are virtually superimposable. These data indicate that the activation of these two serine/threonine kinases by NGF is mediated solely by binding to and activating the pp140c-trk receptor.
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PMID:Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. 132 11

Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
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PMID:The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing. 132 30

Nuclei from bovine thymus contain a high level of partially latent protein phosphatase 1 (PP-1). More than 90% of this PP-1 is associated with the insoluble chromatin/matrix fraction and can be extracted with 0.3 M NaCl. The salt extract also contains three heat- and acid-stable inhibitory proteins of PP-1 that can be resolved on Mono Q. We have purified two of these nuclear inhibitors of PP-1 (NIPP-1a and NIPP-1b) until homogeneity. They are acidic proteins (pI = 4.4) with a molecular mass of 18 kDa (NIPP-1a) and 16 kDa (NIPP-1b) on SDS-PAGE. Judged from the larger molecular mass that was deduced from gel filtration (35 kDa), NIPP-1a and NIPP-1b appear to be asymmetric or dimeric proteins. The nuclear inhibitors totally inhibited the phosphorylase phosphatase activity of PP-1, but even at a 250-fold higher concentration they did not affect the activities of the other major serine/threonine protein phosphatases (PP-2A, PP-2B, and PP-2C). NIPP-1a and NIPP-1b inhibited the catalytic subunit of PP-1 with an extrapolated Ki of about 1 pM, which is some three orders of magnitude better than the cytoplasmic proteins inhibitor 1/DARPP-32 and modulator. The nuclear inhibitors were not inactivated by incubation with protein phosphatases that inactivate inhibitor 1 and DARPP-32. Unlike modulator, they were not able to convert the catalytic subunit of PP-1 into a MgATP-dependent form. Remarkably, the extent of inhibition of PP-1 by NIPP-1b depended on the nature of the substrate. The phosphorylase phosphatase and casein phosphatase activities of PP-1 were completely blocked by NIPP-1b, whereas the dephosphorylation of basic proteins was either not at all inhibited (histone IIA) or only partially (myelin basic protein). These data may indicate that the acidic NIPP-1b is inactivated through complexation by basic proteins. Indeed, nonphosphorylated histone IIA antagonized the inhibitory effect of NIPP-1b on the casein phosphatase activity of PP-1. Our data show that the nucleus contains specific and potent inhibitory proteins of PP-1 that differ from earlier described cytoplasmic inhibitors. We suggest that these novel proteins may control the activity of nuclear PP-1 on its natural substrate(s).
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PMID:The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei. 132 7

Sequences encoding the UL1 gene of equine herpesvirus type 1 (EHV-1) are conserved in the genome of EHV-1 defective interfering particles (DIPs) that mediate oncogenic transformation and persistent infection. The UL1 protein was identified by in vitro transcription/translation and hybrid-arrest translation analyses which employed a UL1/pGEM-3Z construct designated pGEML1. SDS-PAGE analyses of in vitro translation products synthesized from UL1-specific RNA revealed that the UL1 ORF encodes a 30 kDa protein which corresponds in size to the 258 amino acid protein predicted by DNA sequence analyses. This result was confirmed by arresting translation of the in vitro transcribed UL1 RNA with an oligodeoxynucleotide complementary to UL1 coding sequences. The UL1 protein is a homolog of the predicted protein encoded by the ORF2 gene of varicella-zoster virus, but UL1 has no homolog in herpes simplex virus type 1. The UL1 protein contains a region conforming to a 'PEST' (Proline, Glutamic acid, Serine, and Threonine) sequence, which is commonly found in proteins with half-lives of less than two hours.
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PMID:Identification and expression of the UL1 gene product of equine herpesvirus 1. 132 72


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