Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immune interferon-gamma (HuIFN-gamma) labeled with 32P was used to study the structure of IFN-gamma receptor. When [32P]HuIFN-gamma was bound and crosslinked to IFN-gamma the receptor of human cells with a bifunctional crosslinker disuccinimidyl suberate (DSS), a single diffused 32P-labeled band corresponding to the IFN-gamma.receptor complex was visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The size of the [32P]-HuIFN-gamma.receptor complex was about 100-120 kD. Separation of crosslinked complex in reducing and nonreducing gels showed no size differences, suggesting the absence of interchain disulfide linkage. However, binding and formation of the crosslinked IFN-gamma. receptor complex on cells was diminished in the presence of the disulfide reducing agent dithiothreitol (DTT). The reduction was DTT-dose-dependent, suggesting that intramolecular disulfides of the receptor are important for binding. Also, [32P]HuIFN-gamma did not bind if cells were pretreated with and then washed free of DTT, suggesting an irreversible reduction of intrachain disulfide bonds, presumably of the receptor. [32P]HuIFN-gamma also specifically binds to human placental membranes. Each placenta has about 170 ng of IFN-gamma receptors. Covalent attachment of [32P]HuIFN-gamma to placental plasma membranes via DSS produced 2 crosslinked complexes with the molecular sizes of 100-120 kD and 60-70 kD. The IFN-gamma.receptor complex of placental membranes was solubilized with NP-40 after DSS treatment and partially purified with immobilized antibody to the carboxyl terminus of IFN-gamma. Treatment of the receptor complex with trypsin and papain was used to demonstrate its differential proteolytic sensitivity.
 ...
PMID:Immune interferon receptor: chemical and enzymatic sensitivity. 246 13

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated 'cytostatic factor, (CSF)'. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20-30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 x 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.
 ...
PMID:Molecular characteristics of cytostatic factors in amphibian egg cytosols. 256 70

Ornitho-kininogen was purified from chicken and duck blood plasmas by a two-stage method using chromatography on columns of S-alkylated papain-Cellulofine and DEAE-5PW. The isolated preparation from chicken plasma gave a single band on SDS-PAGE with or without 2-mercaptoethanol and on disc-PAGE. The molecular weight of ornitho-kininogen was estimated as 74,000 on SDS-PAGE using the Ferguson plot method. Ornitho-kininogen was found to have the similar properties to those of mammalian high molecular weight kininogen (HMWK), in terms of the amino acid composition, molecular weight, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low molecular weight kininogen (LMWK) and rat T-kininogen could be detected in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed-phase HPLC. The primary structure of the isolated kinin was determined as Arg-Pro-Pro-Gly-Phe-Thr-Pro-Leu-Arg. The sequence of this peptide, named ornitho-kinin, was similar to that of bradykinin except for the substitution of Thr-6 and Leu-8 for Ser-6 and Phe-8. The isolated ornitho-kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe-8 by Leu-8. The sequence of residues 1-30 of ornitho-kininogen exhibited 43% identity with that of bovine kininogen.
 ...
PMID:Ornitho-kininogen and ornitho-kinin: isolation, characterization and chemical structure. 260 3

A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methyl-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with horse urinary kallikrein. After incubation with trypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time of plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was found in this nonpoisonous snake. The dissociation constant (Ki) for the papain-inhibitor complex is approximately 1.6 nM.
 ...
PMID:A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen. 263 47

A murine monoclonal IgM antibody recognizes an antigen (Mo3e) found on the surface of human peripheral blood monocytes stimulated with phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide and muramyl dipeptide and blocks the monocyte response to migration inhibitory factor (MIF). We utilized Western blot and immunoprecipitation analyses of whole cell lysates to investigate the biochemical nature of this monocyte antigen. Two distinct bands (75 kDa and 50 kDa) were detected by anti-Mo3e after monocyte lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto nitrocellulose paper. Stimulation of monocytes with PMA resulted in a marked increase in the amount of the 50-kDa species. Immunoprecipitation of Mo3e from lysates of surface-iodinated cells demonstrated one broad band at 55-80 kDa which increased after PMA stimulation. The epitope identified by anti-Mo3e was resistant to 2-mercaptoethanol and heat treatment (100 degrees C/5 min) and was sensitive to trypsin or papain treatment. Two-dimensional SDS-PAGE analysis revealed that the 75-kDa species has an isoelectric point of 7.0 and the 50-kDa species is more acidic with an isoelectric point of 5.3. These results indicate that anti-Mo3e antibody defines unique monocyte proteins that may play a role as suggested by previous studies, in monocyte activation and responsiveness to MIF.
 ...
PMID:Identification of a human monocyte antigen (Mo3e) associated with cellular activation and lymphokine responsiveness. 265 70

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.
 ...
PMID:Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli. 266 52

The phosphoramidon-insensitive endopeptidase-2 in rat renal brush borders was investigated by immunochemical approaches with a rabbit polyclonal antibody raised to the purified enzyme released from the membrane by papain. An immunoaffinity column successfully purified the detergent-solubilized form of endopeptidase-2. This preparation had an apparent subunit Mr of 80,000, and did not show the two subunits, of Mr 80,000 and 74,000, consistently found in the papain-solubilized forms, indicating that the latter resulted from proteolysis by papain. SDS/polyacrylamide-gel electrophoresis of non-reduced samples of the enzyme revealed a band of Mr 220,000, confirming the presence of disulphide-bridged subunits. Treatment with endoglycosidases H and F generated smaller molecular forms, indicating that endopeptidase-2 contained about 30% asparagine-linked carbohydrate and that a few of these oligosaccharide chains were of the high-mannose type. Treatment with phosphatidylinositol-specific phospholipase indicated that the enzyme did not possess a glycolipid membrane anchor. A survey of rat tissues examined immunohistochemically and by immunoblotting revealed that only the kidney and intestinal tract expressed the antigen in significant amounts. Although some weak staining was seen in salivary glands and thyroid, other organs and tissues including brain and spinal cord were negative by both immunochemical techniques. In the kidney the antigen was confined to the lumen of the proximal tubule and was seen mainly in the population of juxtamedullary nephrons. In the gut, luminal staining was observed throughout its whole length, from duodenum to rectum. Excellent cross-reactivity of the antibody with Balb/c mouse tissues was observed. Immunohistochemistry of mouse kidney and gut revealed a distribution identical with that observed in the rat. Immunopurification of the detergent-solubilized mouse kidney antigen showed it to be a protein containing disulphide-linked subunits of Mr 90,000. It possessed endopeptidase-2-like activity, but was more efficient in hydrolysing azo-casein and less efficient in hydrolysing a model substrate than the rat enzyme. The close similarity between rat endopeptidase-2 and mouse meprin is further supported by these results.
 ...
PMID:Proteins of the kidney microvillar membrane. Structural and immunochemical properties of rat endopeptidase-2 and its immunohistochemical localization in tissues of rat and mouse. 269 Aug 25

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity. 1. Two-chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]noradrenaline release and binding to rat brain membranes. 2. Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N-terminal moiety of the heavy chain, called fragment beta 2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C-terminal part of the heavy chain. 3. The heavy chain alone inhibited K+-evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration-response ratio was flat and 10-100-fold higher concentrations were required than with native or reconstituted two-chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two-chain toxin increased it. Binding to rat brain membranes was assessed by competition with 125I-labelled two-chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two-chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000-fold lower affinity than native or reconstituted two-chain toxin. The light chain did not bind to membranes in either test. Our data indicate that (a) the light chain and the N-terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two-chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.
 ...
PMID:Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties. 275 37

Two peptidases, dehydropeptidase-I and aminopeptidase-M were solubilized from rat kidney microsomes by treatment with papain and separated by DE-52 ion exchange chromatography. Each enzyme was further purified by Sephacryl S-300 gel filtration and affinity chromatography on Con-A Sepharose. Purified dehydropeptidase-I and aminopeptidase-M were homogeneous by SDS-polyacrylamide gel electrophoresis, and their molecular weights were estimated by gel filtration to be 148,000 and 240,000, respectively; both being homodimer, with a 78,000 subunit for the former and a 120,000 subunit for the latter. Both dehydropeptidase-I and aminopeptidase-M were capable of hydrolyzing L-leucyl-L-leucine with a Km valve of 1.1 mM and 1.7 mM, respectively, although the hydrolyzing activity of aminopeptidase-M was much higher than that of dehydropeptidase-I. Aminopeptidase-M was inhibited by bestatin, and dehydropeptidase-I was significantly inhibited by cilastatin. Dehydropeptidase-I catalyzed the conversion of leukotriene D4 to E4 and the hydrolysis of L-cystinyl-bis-glycine, but aminopeptidase-M did not to any appreciable extent. The physiological significance of dehydropeptidase-I was pointed out and discussed.
 ...
PMID:Simultaneous purification and properties of dehydropeptidase-I and aminopeptidase-M from rat kidney. 286 78

We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.
 ...
PMID:Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica. 289 37


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>