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

Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.
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PMID:Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract. 170 70

The heterodimeric vitronectin receptor (VNR) and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) are two members of the integrin family of cell adhesion receptors that share the same beta subunit (GPIIIa). These proteins are involved in binding to vitronectin, fibrinogen and fibronectin and in cytoskeleton-membrane interactions. The present study shows that the human placental syncytiotrophoblast brush border membrane contains a heterodimer of subunit Mr values of 140,000 and 90,000 (non-reduced) or 125,000 and 100,000 (reduced). This protein was recognized by a monoclonal antibody to GPIIIa, rabbit antisera to the VNR and a human alloantiserum to GPIIIa. Brush border VNR-related protein bound to an immobilized peptide containing the Arg-Gly-Asp sequence and, less avidly, to immobilized fibrinogen. Only a small fraction of brush border VNR was associated with a cytoskeleton fraction. Membrane-bound brush border GPIIIa was distinct from that of platelets in its resistance to digestion by trypsin and Staphylococcus aureus V8 protease, and had a slightly lower mobility on SDS/PAGE. In addition, lectin-binding studies indicate glycosylation differences between microvillar and platelet GPIIIa heterodimers. Thus, although placental syncytiotrophoblast expresses a beta 3 integrin in its apical brush border, differences in protease sensitivity and carbohydrate content suggest that it may lack or mask certain antigenic determinants. This may be beneficial in avoiding harmful maternal alloantibody responses during pregnancy. Immunohistology showed that the VNR was present in syncytiotrophoblast apical but not basal plasma membranes, and was absent from other forms of trophoblast. The brush border VNR could function in localizing Arg-Gly-Asp-sequence-containing plasma proteins to the materno-trophoblastic interface.
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PMID:A vitronectin-receptor-related molecule in human placental brush border membranes. 172 Jun 17

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.
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PMID:Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides. 172 59

Cerebrospinal fluid insulin-like growth factor binding proteins (CSF IGFBPs) characteristically have a preferential affinity for IGF-II, which is largely accounted for by a 32-30 kDa IGFBP(Ka = 10(11) M-1) previously purified from human CSF, with an N-terminal sequence unlike any other IGFBP identified at the time. We have now used procedure (including ammonium sulphate precipitation, acidic gel filtration, affinity chromatography and reverse phase chromatography) and purified three further IGFBPs to homogeneity from child CSF. Apart from the 32-30-kDa form, the predominant IGFBP in CSF is a 34-kDa form (non-reduced in SDS-polyacrylamide gel electrophoresis). Like the 32-30-kDa IGFBP, it has a preferential affinity for IGF-II (Ka = 2 x 10(10) M-1). Its N-terminus, Phe-Arg-(/)-Pro-Pro-(/)-Thr-Pro-Glu-Arg-Arg-(/)-Gly-Pro-Pro-Pro-Val, corresponds to that deduced for IGFBP-2, except for the first three residues which were not found in the CSF IGFBP. Another form, of 30 kDa, has an N-terminus identical to IGFBP-3's over the first 18 residues determined and seems to be an altered form of IGFBP-3. A third minor species, of 22 kDa, with a preferential affinity for IGF-II similar to that of the 34-kDa IGFBP, has an N-terminal sequence, (/)-(/)-Asp-Ser-Phe-Val-Pro-(/)-Glu-Pro-Ser-Asp-Glu-Lys-Ala-Leu-Ser-(/)- (/)-Pro, which bears some analogy with those of other known human IGFBPs, particularly IGFBP-3 (7 homologous residues), and appears to be related to, but distinct from, other IGFBP species.
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PMID:Purification from human cerebrospinal fluid of insulin-like growth factor binding proteins (IGFBPs). Isolation of IGFBP-2, an altered form of IGFBP-3 and a new IGFBP species. 172 37

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.
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PMID:Purification and characterization of monodehydroascorbate reductase from soybean root nodules. 172 43

The isolation of cytosol aminopeptidase (CAP) from intestinal mucosa has involved difficult procedures because of low yield, instability of the enzyme and many other factors related to purification. The authors resolved these difficulties in the purification of the enzyme from intestinal mucosa of rats using hydrophobic chromatography on phenyl-TOYOPEARL 650S and ion exchange HPLC on Mono Q 5/5. In this procedure, a 60-fold purification was achieved and the purified enzyme revealed a single band with 56kDa molecular weight by SDS polyacrylamide gel electrophoresis. The purified enzyme preferentially hydrolyzed the Leucyl-containing peptides of Leu-Gly and Leu-Gly-Gly. However, it showed no activity on synthetic substrates of Leu-beta-naphthylamide, Gly-p-nitroanilide, Phe-p-nitroanilide or Met-beta-naphthylamide. The Km and Vmax for Leu-Gly were 0.91 mmol/l and 16.4 mol/min/mg protein, while for Leu-Gly-Gly they were 0.77 mmol/l and 20.6 mol/min/mg protein. The purified enzyme was heat-labile and quickly became less active in highly concentrated ammonium sulfate. The optimum pH was 6.5-10. The enzyme activity was stimulated by Mn2+ and Mg2+, while it was inhibited by EDTA, 1,10-phenanthroline, 2-mercaptoethanol and p-hydroxymercuribenzoate. The results suggested the participation of metal ions and the SH group in the enzyme activity. Furthermore the cytosol aminopeptidase was distinct from the brush-border membrane aminopeptidase in molecular weight, immunoreaction to cytosol aminopeptidase antiserum and the specificities to the substrates.
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PMID:[Purification and characterization of cytosol aminopeptidase from rat intestinal mucosa]. 188 Jan 98

The nematode cuticle is an extracellular structure composed mainly of collagens, with an insoluble epicuticle on the surface. The extracted collagens from adult Ascaris suum can be separated by SDS-PAGE into three major groups of polypeptides with apparent molecular masses of 34, 60-70 and 120-140 kDa. Densitometric evaluation of the polypeptide bands indicated that the three groups are present in the ratio of 1:2:6. Rotary shadowing of reduced, extracted molecules showed fibers 45 nm in length. This length is in excellent agreement with the calculated total length of amino acids in (Gly-X-Y) regions deduced from the collagen gene sequence of Caenorhabditis elegans and A. suum. It is proposed that the three groups of collagen polypeptides found in SDS-PAGE correspond to collagen monomers, dimers and trimers, and that the molecules in the dimeric and trimeric forms are cross-linked via non-reducible bonds.
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PMID:Analysis of the cuticular collagens of Ascaris suum. 197 30

A substitution for a highly conserved non-glycine residue in the triple-helical domain of the pro alpha 2(I) collagen molecule was found in an individual with a variant of the Marfan syndrome. A single base change resulted in substitution of arginine618 by glutamine at the Y position of a Gly-X-Y repeat, and is responsible for the decreased migration in SDS-polyacrylamide gels of some pro alpha 2(I) chains of type I collagen synthesized by dermal fibroblasts from this individual. Family studies suggest that this substitution was inherited from the individual's father who also produces abnormally migrating pro alpha 2(I) collagen chains and shares some of the abnormal skeletal features. This single base change creates a new Bsu36 I (Sau I, Mst II) restriction site detectable in genomic DNA by Southern blot analysis when probed with a COL1A2 fragment. The analysis of 52 control individuals (103 chromosomes) was negative for the new Bsu36 I site, suggesting that the substitution is not a common polymorphism.
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PMID:A substitution at a non-glycine position in the triple-helical domain of pro alpha 2(I) collagen chains present in an individual with a variant of the Marfan syndrome. 197 25

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z-Phe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possessed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase; it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.
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PMID:Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis. 199 77

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.
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PMID:The presence of a covalently cross-linked matrix in human platelets. 200 80


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