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 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

One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 28-kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlamydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those Chlamydomonas ubiquitin [corrected] and wheat histone H2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease V8-digested polypeptide fragments of the 28-kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonas 28-kDa ubiquitinated protein is monoubiquitinated histone H2B.
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PMID:Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B. 131 4

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.
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PMID:Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane. 132 65

We developed three antibodies, specific and sensitive to endothelin-1 (ET-1), and established two sandwich and three competitive enzyme immunoassays (EIAs). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-1 but not with the antibody against C-terminal peptide of ET-1, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-1.
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PMID:Characterization of immunoreactive endothelin in human urine. 154 Jan 94

The clinical and biochemical observations in a patient with a mild form of Ehlers-Danlos syndrome (EDS) type IV are described. The patient's skin fibroblasts produced markedly diminished amounts of type III collagen. SDS-polyacrylamide gel electrophoresis of collagens produced by cells obtained from other, non-cutaneous tissues showed two forms of collagen alpha 1(III) chains, a normal and a slow migrating, mutant form. Further analysis confirmed that the type III collagen molecules containing mutant alpha chains which were overmodified had a lower thermal stability and were poorly secreted into the extracellular medium. The protein defect was mapped by in situ cyanogen bromide digestion and was located in alpha 1(III) CB9, the C-terminal peptide of the collagen triple helix. This study shows that non-cutaneous connective tissues can be a useful source for the study of type III collagen defects in patients with EDS type IV.
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PMID:Detection and characterisation of an overmodified type III collagen by analysis of non-cutaneous connective tissues in a patient with Ehlers-Danlos syndrome IV. 161 32

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.
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PMID:Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane. 171 97

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position 150 following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS-polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH2-terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gln residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.
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PMID:Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts. 179 12

Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.
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PMID:Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing. 201 90

We have reviewed the structure, function, and biogenesis of mammalian cytochrome c oxidase, examined the tissue-specific expression of isoforms of cytochrome c oxidase subunits in different mammals, and attempted to correlate the data with our knowledge of cytochrome c oxidase deficiency, illustrated by one particular patient. Cytochrome c oxidase was isolated from bovine tissues, and individual subunits examined by SDS-PAGE, N-terminal peptide sequencing, and antibody binding. Isoforms of subunits VIa, VIIa, and VIII were identified, manifesting one pattern of expression in heart and skeletal muscle, and another in liver, kidney, and brain. In rat heart and liver, only one form of subunit VIIa was identified. Northern analysis of bovine and rat tissues suggested that the tissue-specific expression of subunits VIa and VIII is regulated transcriptionally in liver, kidney, and brain, and posttranscriptionally in heart and skeletal muscle. In humans, antibody binding documented isoforms of subunits VIa and VIIa, with the pattern of expression in heart and skeletal muscle differing from that in liver, kidney, and brain; our data suggested that both isoforms of subunit VIa may be expressed in human heart. In a patient with cytochrome c oxidase deficiency, the clinical, morphologic, and biochemical manifestations were much more severe in heart than in skeletal muscle. Antibody binding suggested partial assembly of the enzyme in heart. These and other data suggest considerably more variability in the tissue-specific expression of isoforms of cytochrome c oxidase subunits than previously recognized.
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PMID:Isoforms of mammalian cytochrome c oxidase: correlation with human cytochrome c oxidase deficiency. 217 25

Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.
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PMID:Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas. 224 65


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